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Phagocytosis is a specialized form of endocytosis where large cells and particles (>0.5μm) are engulfed by the phagocytic cells, and ultimately digested in the phagolysosomes. This process not only eliminates unwanted particles and pathogens from the extracellular sources, but also eliminates apoptotic cells within the body, and is critical for maintenance of tissue homeostasis. It is believed that both endocytosis and phagocytosis share common pathways after particle internalization, but specialized features and differences between these two routes of internalization are also likely. The recruitment and removal of each protein/particle during the maturation of endocytic/phagocytic vesicles has to be tightly regulated to ensure their timely action. Ubiquitin proteasome pathway (UPP), degrades unwanted proteins by post-translational modification of proteins with chains of conserved protein Ubiquitin (Ub), with subsequent recognition of Ub chains by the 26S proteasomes and substrate degradation by this protease. This pathway utilizes different Ub linkages to modify proteins to regulate protein-protein interaction, localization, and activity. Due to its vast number of targets, it is involved in many cellular pathways, including phagocytosis. This chapters describes the basic steps and signaling in phagocytosis and different roles that UPP plays at multiple steps in regulating phagocytosis directly, or through its interaction with other phagosomal proteins. How aberrations in UPP function affect phagocytosis and their association with human diseases, and how pathogens exploit this pathway for their own benefit is also discussed., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Herein, we provide a colony forming unit (CFU)-based counting method for quantitating the bacterial binding, phagocytosis, and killing capacity of phagocytes. Although these functions can be measured by immunofluorescence- and dye-based assays, quantitating CFUs are comparatively inexpensive and easy to perform. The protocol described below is easily modified for use with different phagocytes (e.g., macrophages, neutrophils, cell lines), types of bacteria, or opsonic conditions., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Following phagocytosis, the nascent phagosome undergoes maturation to become a phagolysosome with an acidic, hydrolytic, and often oxidative lumen that can efficiently kill and digest engulfed microbes, cells, and debris. The fusion of phagosomes with lysosomes is a principal driver of phagosomal maturation and is targeted by several adapted intracellular pathogens. Impairment of this process has significant consequences for microbial infection, tissue inflammation, the onset of adaptive immunity, and disease. Given the importance of phagosome-lysosome fusion to phagocyte function and the many virulence factors that target it, it is unsurprising that multiple molecular pathways have evolved to mediate this essential process. While the full range of these pathways has yet to be fully characterized, several pathways involving proteins such as members of the Rab GTPases, tethering factors and SNAREs have been identified. Here, we summarize the current state of knowledge to clarify the ambiguities in the field and construct a more comprehensive phagolysosome formation model. Lastly, we discuss how other cellular pathways help support phagolysosome biogenesis and, consequently, phagocyte function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nguyen and Yates.)

The membrane attack complex (MAC) of the complement system and Perforin-1 are well characterized innate immune effectors. MAC is composed of C9 and other complement proteins that target the envelope of gram-negative bacteria. Perforin-1 is deployed when killer lymphocytes degranulate to destroy virally infected or cancerous cells. These molecules polymerize with MAC-perforin/cholesterol-dependent cytolysin (MACPF/CDC) domains of each monomer deploying amphipathic β-strands to form pores through target lipid bilayers. In this review we discuss one of the most recently discovered members of this family; Perforin-2, the product of the Mpeg1 gene. Since their initial description more than 100 years ago, innumerable studies have made macrophages and other phagocytes some of the best understood cells of the immune system. Yet remarkably it was only recently revealed that Perforin-2 underpins a pivotal function of phagocytes; the destruction of phagocytosed microbes. Several studies have established that phagocytosed bacteria persist and in some cases flourish within phagocytes that lack Perforin-2. When challenged with either gram-negative or gram-positive pathogens Mpeg1 knockout mice succumb to infectious doses that the majority of wild-type mice survive. As expected by their immunocompromised phenotype, bacterial pathogens replicate and disseminate to deeper tissues of Mpeg1 knockout mice. Thus, this evolutionarily ancient gene endows phagocytes with potent bactericidal capability across taxa spanning sponges to humans. The recently elucidated structures of mammalian Perforin-2 reveal it to be a homopolymer that depends upon low pH, such as within phagosomes, to transition to its membrane-spanning pore conformation. Clinical manifestations of Mpeg1 missense mutations further highlight the pivotal role of Perforin-2 within phagocytes. Controversies and gaps within the field of Perforin-2 research are also discussed as well as animal models that may be used to resolve the outstanding issues. Our review concludes with a discussion of bacterial counter measures against Perforin-2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Merselis, Rivas and Munson.)

Calcium (Ca 2+ ) is a ubiquitous second messenger involved in the regulation of numerous cellular functions including vesicular trafficking, cytoskeletal rearrangements and gene transcription. Both global as well as localized Ca 2+ signals occur during phagocytosis, although their functional impact on the phagocytic process has been debated. After nearly 40 years of research, a consensus may now be reached that although not strictly required, Ca 2+ signals render phagocytic ingestion and phagosome maturation more efficient, and their manipulation make an attractive avenue for therapeutic interventions. In the last decade many efforts have been made to identify the channels and regulators involved in generating and shaping phagocytic Ca 2+ signals. While molecules involved in store-operated calcium entry (SOCE) of the STIM and ORAI family have taken center stage, members of the canonical, melastatin, mucolipin and vanilloid transient receptor potential (TRP), as well as purinergic P2X receptor families are now recognized to play significant roles. In this chapter, we review the recent literature on research that has linked specific Ca 2+ -permeable channels and regulators to phagocytic function. We highlight the fact that lipid mediators are emerging as important regulators of channel gating and that phagosomal ionic homeostasis and Ca 2+ release also play essential parts. We predict that improved methodologies for measuring these factors will be critical for future advances in dissecting the intricate biology of this fascinating immune process.

Atopic bronchial asthma based on allergy history and chronic inflammation is hazardous to patients due to the risk of exacerbation. The sign of severe exacerbation is considered an abundant number and high activity of granulocytes in respiratory system and blood. Relationships between the ability of cells in blood to produce reactive radicals and their metabolites and the severity of asthma remain largely unclear. Kinetics of respiratory burst evoked by microbe particles in blood samples of patients was studied to reveal the most significant predictors distinguishing states of moderate exacerbation and out of exacerbation. Asthmatic patients with exacerbation (n = 18) or out of exacerbation (n = 62) and healthy individuals (n = 43) were characterized on respiratory function, cell count in blood and kinetics of generation of reactive radicals and their metabolites during phagocytosis. Mean values of respiratory parameters forced expiratory volume in 1 s and peak expiratory flow rate in patients with exacerbation were significantly differed compared with same of patients out of exacerbation and healthy individuals. Mean values of cell count in blood did not significantly differed in patients with exacerbation and out of exacerbation. Receiver operating characteristic analysis showed that both cell count and respiratory indexes did not discriminate patients with exacerbation from out of exacerbation. A delayed response to opsonized zymosan was revealed in patients with exacerbation compared to other examinees: lengthened lag-time and T max , reduced production of reactive species. T max was the most statistically significant predictor to discriminate bronchial asthma exacerbation from bronchial asthma out of exacerbation (area under curve >90%, p < 10 -5 ) and controls (area under curve >80%, p < 10 -5 ). Thus kinetic parameters of the phagocyte response to opsonized zymosan in the whole blood are the best predictors of bronchial asthma exacerbation in comparison with respiratory parameters and blood cell count. This test can be used for immunological monitoring of bronchial asthma status to prevent exacerbation., (Copyright © 2019. Published by Elsevier Inc.)

In advanced atherosclerotic plaques, defective efferocytosis of apoptotic foam cells and decreased cholesterol efflux contribute to lesion progression. In our previous study, we demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (SDT) could induce foam cells apoptosis via the mitochondrial-caspase pathway. In the current research, we sought to explore ALA-SDT-induced apoptosis of phagocytes and the effects of cholesterol efflux and efferocytosis in advanced apoE -/- mice plaque. Methods: apoE -/- mice fed western diet were treated with ALA-SDT and sacrificed at day 1, day 3, day 7 and day 28 post treatment. THP-1 macrophage-derived foam cells were treated with ALA-SDT. 5 hours later, the supernatant was collected and added to fresh foam cells (phagocytes). Then, the lipid area, efferocytosis, cholesterol efflux, anti-inflammatory reactions and PPARγ-LXRα-ABCA1/ABCG1 pathway were detected in plaque in vivo and in phagocytes in vitro . Results: We found that ALA-SDT induced foam cells apoptosis coupled with efferocytosis and upregulation of Mer tyrosine kinase (MerTK) both in vivo and in vitro . The lipid content in plaque decreased as early as 1 day after ALA-SDT and this tendency persisted until 28 days. The enhancement of phagocytes cholesterol efflux was accompanied by an approximately 40% decrease in free cholesterol and a 24% decrease in total cholesterol in vitro . More importantly, anti-inflammatory factors such as TGFβ and IL-10 were upregulated by ALA-SDT treatment. Finally, we found that PPARγ-LXRα-ABCA1/ABCG1 pathway was activated both in vivo and in vitro by ALA-SDT, which could be blocked by PPARγ siRNA. Conclusions: Activation of PPARγ-LXRα-ABCA1/ABCG1 pathway induced by ALA-SDT treatment engages a virtuous cycle that enhances efferocytosis, cholesterol efflux and anti-inflammatory reactions in advanced plaque in vivo and in phagocytes in vitro ., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation-reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher ( p  < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity.

Background: Microglial cells (MCs) are the sentries of the central nervous system. In health, they are known as surveying MCs because they examine the tissue to maintain the homeostasis. In disease, they activate and, among other functions, become phagocytic to clean the cellular debris. In this work, we have studied the behavior of rat retinal MCs in two models of unilateral complete intraorbital optic nerve axotomy which elicit a different time course of retinal ganglion cell (RGC) loss., Methods: Albino Sprague-Dawley rats were divided into these groups: (a) intact (no surgery), (b) fluorogold (FG) tracing from the superior colliculi, and (c) FG tracing + crush or transection of the left optic nerve. The retinas were dissected from 2 days to 2 months after the lesions (n = 4-12 group/lesion and time point) and then were subjected to Brn3a and Iba1 double immunodetection. In each intact retina, the total number of Brn3a + RGCs and Iba + MCs was quantified. In each traced retina (b and c groups), FG-traced RGCs and phagocytic microglial cells (PMCs, FG + Iba + ) were also quantified. Topographical distribution was assessed by neighbor maps., Results: In intact retinas, surveying MCs are homogenously distributed in the ganglion cell layer and the inner plexiform layer. Independently of the axotomy model, RGC death occurs in two phases, one quick and one protracted, and there is a lineal and topographical correlation between the appearance of PMCs and the loss of traced RGCs. Furthermore, the clearance of FG + RGCs by PMCs occurs 3 days after the actual loss of Brn3a expression that marks RGC death. In addition, almost 50% of MCs from the inner plexiform layer migrate to the ganglion cell layer during the quick phase of RGC loss, returning to the inner plexiform layer during the slow degeneration phase. Finally, in contrast to what happens in mice, in rats, there is no microglial phagocytosis in the contralateral uninjured retina., Conclusions: Axotomy-induced RGC death occurs earlier than RGC clearance and there is an inverse correlation between RGC loss and PMC appearance, both numerically and topographically, suggesting that phagocytosis occurs as a direct response to RGC death rather than to axonal damage.

In this study, we evaluated the inhibitory effect of a rice bran mixture extract (RBE) on Brucella abortus pathogenesis in professional (RAW 264.7) and nonprofessional (HeLa) phagocytes. We fermented the rice bran mixture and then extracted it with 50% ethanol followed by gas chromatography-mass spectrometry to identify the components in RBE. Our results clearly showed that RBE caused a significant reduction in the adherence of B. abortus in both cell lines. Furthermore, analysis of phagocytic signaling proteins by western blot assay revealed that RBE pretreatment resulted in inhibition of phosphorylation of JNK, ERK, and p38, leading to decline of internalization compared with the controls. Additionally, the intensity of F-actin observed by fluorescence microscopy and FACS was remarkably reduced in RBE-pretreated cells compared with control cells. However, the intracellular replication of B. abortus within phagocytes was not affected by RBE. Taken together, these findings suggest that the phagocytic receptor blocking and suppressive effects of RBE on the MAPK-linked phagocytic signaling pathway could negatively affect the invasion of B. abortus into phagocytes.

Phagocytosis, a critical early event in the microbicidal response of neutrophils, is now appreciated to serve multiple functions in a variety of cell types. Professional phagocytes play a central role in innate immunity by eliminating pathogenic bacteria, fungi and malignant cells, and contribute to adaptive immunity by presenting antigens to lymphocytes. In addition, phagocytes play a part in tissue remodeling and maintain overall homeostasis by disposing of apoptotic cells, a task shared by non-professional phagocytes, often of epithelial origin. This functional versatility is supported by a vast array of receptors capable of recognizing a striking variety of foreign and endogenous ligands. Here we present an abbreviated overview of the different types of phagocytes, their varied modes of signaling and particle engulfment, and the multiple physiological roles of phagocytosis.

Herein we provide a colony forming unit (CFU)-based counting method for quantitating the bacterial binding, phagocytosis, and killing capacity of phagocytes. Although these functions can be measured by immunofluorescence and dye-based assays, quantitating CFUs is comparatively inexpensive and easy to perform. The protocol described below is easily modified for use with different phagocytes (e.g., macrophages, neutrophils, cell lines), types of bacteria or opsonic conditions.

Phagocytosis is a critical biological activity through which the host can protect itself from infectious and non-infectious environmental particles and remove unwanted host cells in order to maintain tissue homeostasis. Phagocytosis is an ancient, conserved process that is apparent in all multicellular organisms. The process of phagocytosis requires the recognition of ligands on particles by specific receptors expressed by the phagocyte that promote internalization via reorganization of cytoskeletal elements and directed formation of the phagosome. Subsequent phagosome-lysosome fusion delivers the contents for destruction and recycling in the acidic compartment. Significantly, receptor engagement and uptake can also trigger intracellular signaling pathways that initiate appropriate innate immune and pro-inflammatory or anti-inflammatory responses dependent upon the nature of the particle. The important benefits of phagocytosis to host survival are exemplified by the detrimental effects to health that occur when phagocytic efficiency is diminished. In an overview, we discuss the different experimental approaches or options that can be considered when investigating and determining the characteristics and quantification of phagocytic activity. These criteria will include choice of phagocytic cell type, selection, and method of labeling of particle for monitoring internalization, targeting of particles to specific receptors, and quantification of ingestion either at the single cell or at the population level. We provide two detailed examples of phagocytosis assays., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidification is required for many enzymatic processes taking place in phago-lysosomes, like proteolysis, and supports the microbicidal activity of macrophages. Here we present a new quantitative methodology to assess phagosome acidification by flow cytometry based on the use of bi-fluorescent particles. This method relies on the use of UV polystyrene beads labelled with the acid sensor pHrodo-succinimidyl ester (pHrodo(TM) SE red) and enables us to dissociate particle association with phagocytes from their engulfment in acidified compartments. This methodology is well suited to monitor the acidification of phagosomes formed in vivo after fluorescent bead administration., (Copyright © 2014. Published by Elsevier B.V.)

Cysteine cathepsins are endolysosomal cysteine proteases highly expressed in macrophages; however, their individual contributions to the elimination of bacteria and bacteria-induced cytokine production by macrophages are unknown. We assessed the contribution of cysteine cathepsins to macrophage defense pathways against Staphylococcus aureus by using chemical inhibitors and by infecting primary bone marrow-derived macrophages deficient in 1 of 7 major macrophage-expressed endolysosomal cysteine proteases. We show that cysteine cathepsins are involved in the phagocytosis and killing of S. aureus. Cathepsin L was identified as an executor of nonoxidative killing. Moreover, microarray data revealed cysteine cathepsins to be important for the maximal induction of certain proinflammatory genes, such as IL6, in response to S. aureus. Cysteine cathepsin's contribution to IL6 production was dependent on phagocytosis, and cathepsin K was identified to be a critical protease in this process. Analysis of macrophages with impaired trafficking of endolysosomal Toll-like receptors (TLRs) to the acidic compartment revealed that they were not involved in cathepsin-dependent IL6 induction. Because IL6 production was completely dependent on the TLR-adaptor protein myeloid differentiation primary response gene 88 (MyD88), it appears that other TLRs are involved. In summary, lysosomal cysteine proteases are functionally linked to the complex bactericidal and inflammatory activities of macrophages.

Microcystin-LR (MC-LR) is the main isoform of hepatotoxin produced by cyanobacteria, which occur worldwide in the aquatic environment. The present study investigated the in vitro toxic MC-LR effects on immune cells isolated from the blood of carp. Cells were exposed to different MC-LR concentrations ranging from 0.01 to 1 µg ml(-1) for 2, 6 and 24 h. In addition, the effect of the toxin on the phagocytic activity of leukocytes and on actin and tubulin re-organization in phagocytic cells was studied. We observed that MC-LR induces apoptosis in lymphocytes 2 h after incubation, whereas high toxin concentrations induced necrosis in lymphocytes in a time- and concentration-dependent manner. Incubation of the cells for 2 h with 0.1 and 1 µg ml(-1) MC-LR inhibited phagocytosis without affecting apoptosis or glutathione (GSH) levels. Moreover, at this time point and with these concentrations, the toxin also induced a significant re-organization of the actin cytoskeleton in phagocytes, which subsequently collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. These results suggest that both phagocytes and lymphocytes are targets for MC-LR and the disturbances of phagocytosis may impair the balance of the immune system., (Copyright © 2012 John Wiley & Sons, Ltd.)

Introduction: Blood-feeding arthropods rely on robust cellular and humoral immunity to control pathogen invasion and replication. Tick hemocytes produce factors that can facilitate or suppress microbial infection and pathogenesis. Despite the importance of hemocytes in regulating microbial infection, understanding of their basic biology and molecular mechanisms remains limited. Methods: Here we combined histomorphology and functional analysis to identify five distinct phagocytic and non-phagocytic hemocyte populations circulating within the Gulf Coast tick Amblyomma maculatum. Results and discussion: Depletion of phagocytic hemocytes using clodronate liposomes revealed their function in eliminating bacterial infection. We provide the first direct evidence that an intracellular tick-borne pathogen, Rickettsia parkeri, infects phagocytic hemocytes in Am. maculatum to modify tick cellular immune responses. A hemocyte-specific RNA-seq dataset generated from hemocytes isolated from uninfected and R. parkeri-infected partially blood-fed ticks generated ~40,000 differentially regulated transcripts, >11,000 of which were immune genes. Silencing two differentially regulated phagocytic immune marker genes (nimrod B2 and eater-two Drosophila homologs), significantly reduced hemocyte phagocytosis. Conclusion: Together, these findings represent a significant step forward in understanding how hemocytes regulate microbial homeostasis and vector competence. [ABSTRACT FROM AUTHOR]

This work seeks to establish a theoretical model that links phagocytic capacity of phagocytes with geometries of phagocytes and phagocytic objects. The model is applied to four different types of phagocytic objects: a flat surface, a microsphere, a microdisk, and a population of microspheres. For the flat surface, the model assumes that a phagocyte maximizes its contact area between the flat surface and minimizes its non‐contact surface area under a constraint exerted by the volume of the phagocyte's cellular contents. For any of the other three types of objects, the model assumes that a phagocyte completely phagocytoses the object(s) by exhausting its available membrane area. It also assumes that the post‐phagocytosis phagocyte minimizes its surface area. Moreover, the volume of the phagocyte's cellular contents and the geometry of the object impose constraints on the model. For each case, two governing equations are established and combined to derive a single explicit equation with the phagocyte's phagocytic capacity as a function of other parameters. The equations are applied to experimental data for macrophages and neutrophils in the literature. Methods for extending the model and experimentally testing it are discussed. [ABSTRACT FROM AUTHOR]

The last step of cell death is cell clearance, a process critical for tissue homeostasis. For efficient cell clearance to occur, phagocytes and dead cells need to reciprocally signal to each other. One important phenomenon that is under-investigated, however, is that phagocytes not only engulf corpses but contribute to cell death progression. The aims of this study were to determine how the phagocytic receptor Draper non-autonomously induces cell death, using the Drosophila ovary as a model system. We found that Draper, expressed in epithelial follicle cells, requires its intracellular signaling domain to kill the adjacent nurse cell population. Kinases Src42A, Shark and JNK (Bsk) were required for Draper-induced nurse cell death. Signs of nurse cell death occurred prior to apparent engulfment and required the caspase Dcp-1, indicating that it uses a similar apoptotic pathway to starvation-induced cell death. These findings indicate that active signaling by Draper is required to kill nurse cells via the caspase Dcp-1, providing novel insights into mechanisms of phagoptosis driven by non-professional phagocytes. [ABSTRACT FROM AUTHOR]

LC3-associated phagocytosis (LAP) is a noncanonical autophagy process reported in recent years and is one of the effective mechanisms of host defense against bacterial infection. During LAP, bacteria are recognized by pattern recognition receptors (PRRs), enter the body, and then recruit LC3 onto a single-membrane phagosome to form a LAPosome. LC3 conjugation can promote the fusion of the LAPosomes with lysosomes, resulting in their maturation into phagolysosomes, which can effectively kill the identified pathogens. However, to survive in host cells, bacteria have also evolved strategies to evade killing by LAP. In this review, we summarized the mechanism of LAP in resistance to bacterial infection and the ways in which bacteria escape LAP. We aim to provide new clues for developing novel therapeutic strategies for bacterial infectious diseases. [ABSTRACT FROM AUTHOR]

Phagocytes, such as macrophages and dendritic cells, possess the ability to ingest large quantities of exogenous material into membrane-bound endocytic organelles such as macropinosomes and phagosomes. Typically, the ingested material, which consists of diverse macromolecules such as proteins and nucleic acids, is delivered to lysosomes where it is digested into smaller molecules like amino acids and nucleosides. These smaller molecules can then be exported out of the lysosomes by transmembrane transporters for incorporation into the cell's metabolic pathways or for export from the cell. There are, however, exceptional instances when undigested macromolecules escape degradation and are instead delivered across the membrane of endocytic organelles into the cytosol of the phagocyte. For example, double stranded DNA, a damage associated molecular pattern shed by necrotic tumor cells, is endocytosed by phagocytes in the tumor microenvironment and delivered to the cytosol for detection by the cytosolic "danger" sensor cGAS. Other macromolecular "danger" signals including lipopolysaccharide, intact proteins, and peptidoglycans can also be actively transferred from within endocytic organelles to the cytosol. Despite the obvious biological importance of these processes, we know relatively little of how macromolecular "danger" signals are transferred across endocytic organelle membranes for detection by cytosolic sensors. Here we review the emerging evidence for the active cytosolic transfer of diverse macromolecular "danger" signals across endocytic organelle membranes. We will highlight developing trends and discuss the potential molecular mechanisms driving this emerging phenomenon. [ABSTRACT FROM AUTHOR]

Humans are exposed to numerous synthetic foreign particles in the form of drug delivery systems and diagnostic agents. Specialized immune cells (phagocytes) clear these particles by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particles may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize Escherichia coli (E. coli) faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and Staphylococcus epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable abiotic and biotic material. [Display omitted] • Uptake of particulate matter drives sequential phagocytosis in macrophages, monocytes, and neutrophils. • Increased sequential phagocytosis is not a result of Toll-like receptor driven activation or cellular heterogeneity. • Sequential phagocytosis is likely driven by changes to the fluidity of the cell membrane and cellular compliance. • One application of the enhanced phagocytosis driven by particulates is to increase the clearance rates of bacteria. [ABSTRACT FROM AUTHOR]

Macrophage activation in the presence of bacterial cells and molecules entails complex programs of gene expression. How such triggers elicit specific gene expression programs is incompletely understood. We previously discovered that TFEB (transcription factor EB) is a key contributor to macrophage activation during bacterial phagocytosis. However, the mechanism linking phagocytosis of bacterial cells to TFEB activation and downstream pro-inflammatory cytokine induction remained unknown. We found that macrophages lacking both TFEB and TFE3 (transcription factor E3) were unable to mount a pro-inflammatory phenotype in response to bacterial infection. The NOX/PHOX (NADPH oxidase)-dependent oxidative burst was required for nuclear translocation of TFEB during phagocytosis of Gram-positive or -negative bacteria, and reactive oxygen species (ROS) were sufficient to trigger TFEB activation in a CD38- and NAADP (nicotinic acid adenine dinucleotide phosphate)-dependent manner. Consistent with the Ca2+-releasing activity of NAADP, intracellular Ca2+ chelation and PPP3/calcineurin inhibition prevented TFEB activation by phagocytosis and ROS (reactive oxygen species), impairing the induction of pro-inflammatory cytokines such as IL6 and TNF/TNFα. Therefore, here we describe a previously unknown pathway that links phagocytosis with macrophage pro-inflammatory polarization via TFEB and related transcription factor TFE3. These findings reveal that activation of TFEB and TFE3 is a key regulatory event for the activation of macrophages, and have important implications for infections, inflammation, cancer, obesity, and atherosclerosis. [ABSTRACT FROM AUTHOR]

How phagocytes find invading microorganisms and eliminate pathogenic ones from human bodies is a fundamental question in the study of infectious diseases. About 2.5 billion years ago, eukaryotic unicellular organisms–protozoans–appeared and started to interact with various bacteria. Less than 1 billion years ago, multicellular animals–metazoans–appeared and acquired the ability to distinguish self from non-self and to remove harmful organisms from their bodies. Since then, animals have developed innate immunity in which specialized white-blood cells phagocytes- patrol the body to kill pathogenic bacteria. The social amoebae Dictyostelium discoideum are prototypical phagocytes that chase various bacteria via chemotaxis and consume them as food via phagocytosis. Studies of this genetically amendable organism have revealed evolutionarily conserved mechanisms underlying chemotaxis and phagocytosis and shed light on studies of phagocytes in mammals. In this review, we briefly summarize important studies that contribute to our current understanding of how phagocytes effectively find and kill pathogens via chemotaxis and phagocytosis.

Canine parvoviral enteritis (CPE) is a severe disease characterized by systemic inflammation and immunosuppression. The function of circulating phagocytes (neutrophils and monocytes) in affected dogs has not been fully investigated. We characterized the functional capacity of canine phagocytes in CPE by determining their oxidative burst and phagocytic activities using flow cytometry. Blood was collected from 28 dogs with CPE and 11 healthy, age-matched, control dogs. Oxidative burst activity was assessed by stimulating phagocytes with opsonized Escherichia coli or phorbol 12-myristate 13-acetate (PMA) and measuring the percentage of phagocytes producing reactive oxygen species and the magnitude of this production. Phagocytosis was measured by incubating phagocytes with opsonized E. coli and measuring the percentage of phagocytes containing E. coli and the number of bacteria per cell. Complete blood counts and serum C-reactive protein (CRP) concentrations were also determined. Serum CRP concentration was negatively and positively correlated with segmented and band neutrophil concentrations, respectively. Overall, no differences in phagocyte function were found between dogs with CPE and healthy control dogs. However, infected dogs with neutropenia or circulating band neutrophils had decreased PMA-stimulated oxidative burst activity compared to healthy controls. Additionally, CPE dogs with neutropenia or circulating band neutrophils had decreased PMA- and E. coli –stimulated oxidative burst activity and decreased phagocytosis of E. coli compared to CPE dogs without neutropenia or band neutrophils. We conclude that phagocytes have decreased oxidative burst and phagocytic activity in neutropenic CPE dogs and in CPE dogs with circulating band neutrophils. [ABSTRACT FROM AUTHOR]

Macrophage hyperfunction or dysfunction is tightly associated with various diseases, such as osteoporosis, inflammatory disorder, and cancers. However, nearly all conventional drug delivery system (DDS) nanocarriers utilize endocytosis for entering target cells; thus, the development of macrophage-targeting and phagocytosis-inducing DDS nanocarriers for treating these diseases is required. In this study, we developed a hepatitis B virus (HBV) envelope L particle ( i.e. , bio-nanocapsule (BNC)) outwardly displaying a tandem form of protein G-derived IgG Fc-binding domain and protein L-derived IgG Fab-binding domain (GL-BNC). When conjugated with the macrophage-targeting ligand, mouse IgG2a (mIgG2a), the GL-BNC itself, and the liposome-fused GL-BNC ( i.e. , GL-virosome) spontaneously initiated aggregation by bridging between the Fc-binding domain and Fab-binding domain with mIgG2a. The aggregates were efficiently taken up by macrophages, whereas this was inhibited by latrunculin B, a phagocytosis-specific inhibitor. The mIgG2a-GL-virosome containing doxorubicin exhibited higher cytotoxicity toward macrophages than conventional liposomes and other BNC-based virosomes. Thus, GL-BNCs and GL-virosomes may constitute promising macrophage-targeting and phagocytosis-inducing DDS nanocarriers. Statement of Significance We have developed a novel macrophage-targeting and phagocytosis-inducing bio-nanocapsule (BNC)-based nanocarrier named GL-BNC, which comprises a hepatitis B virus envelope L particle outwardly displaying protein G-derived IgG Fc- and protein L-derived IgG Fab-binding domains in tandem. The GL-BNC alone or liposome-fused form (GL-virosomes) could spontaneously aggregate when conjugated with macrophage-targeting IgGs, inducing phagocytosis by the interaction between IgG Fc of aggregates and FcγR on phagocytes. Thereby these aggregates were efficiently taken up by macrophages. GL-virosomes containing doxorubicin exhibited higher cytotoxicity towards macrophages than ZZ-virosomes and liposomes. Our results suggested that GL-BNCs and GL-virosomes would serve as promising drug delivery system nanocarriers for targeting delivery to macrophages. [ABSTRACT FROM AUTHOR]

Our understanding of the humoral immune response in both vertebrates and invertebrates has dramatically deepened in the past decade. In contrast, many of the mechanisms and roles of the cellular immune response remain to be elucidated. Phagocytosis is at the center of the cellular responses in both innate and adaptive immunity. Targets of phagocytosis are either invading microbes or altered self, that is, own cells that have become dispensable or harmful. The selective recognition and engulfment of target cells by phagocytes are achieved through the specific binding of receptors of phagocytes to ligands present on the surface of the target cells. However, these phagocytosis receptors and ligands are still being identified. The fundamental mechanism of phagocytosis appears to be the same in vertebrates and invertebrates, but whether or not genes are evolutionally conserved has yet to be determined.

While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth.We employed different models of M1 and M2 polarization: GM-CSF/LPS/IFN-gamma was used to generate primary human M1 cells and M-CSF/IL-10 to differentiate M2 monocyte-derived macrophages. PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-gamma and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø 26 and 41 nm) and microparticles (Ø 1.75 µm) was quantified. At the concentration used (50 µg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human monocyte-derived macrophages compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages (TAM) obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue.In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but might also open up therapeutic perspectives allowing to specifically target M2 polarized macrophages.