Mechanism Of Phagocytosis Research Articles

Susceptibility to malaria in fulani, Bariba, Otamari and gando individuals living in sympatry in Benin: Role of opsonizing antibodies to Plasmodium falciparum merozoites

ObjectivesFulani in Africa are known to be less susceptible to Plasmodium falciparum (Pf) malaria. This study explored a potential involvement of antibody-mediated merozoite phagocytosis mechanism in this natural protection against malaria. MethodsBefore the start of the malaria transmission season (MTS) in Benin, the functionality of antibodies against Pf merozoites was determined by the opsonic phagocytosis (OP) assay in plasma samples from Fulani, Bariba, Otamari and Gando groups. These individuals were actively followed-up for malaria detection from the beginning to the end of MTS. Anti-GLURP Immunoglobulin G antibody quantification, malaria Rapid Diagnostic Test (RDT) and spleen palpation were performed before and after MTS. ResultsIn Bariba, Otamari and Gando, but not in Fulani, plasma from adults promoted higher levels of OP than the children (P = 0.003; P = 0.012; P = 0.031 and P = 0.122). A high proportion of Fulani children had higher OP and anti-GLURP (P < 0.0001) antibody levels as compared to non-Fulani children; whereas this was not observed for Fulani adults (P = 0.223). High OP levels before MTS were significantly related to negative RDT after MTS (P = 0.011). ConclusionOur results highlight the ability of opsonizing antibodies to potentially enhance natural protection of young Fulani individuals against Pf malaria in Benin.

STATE OF THE PHAGOCYTIC LINK OF IMMUNITY IN CHOLANGITIS

Summary. Aim. To study the condition of the phagocytic part of immunity in cholangitis. One of the main ways of elimination of bacterial contamination of the body is the work of the immune system, namely phagocytosis. This especially applies to acute and chronic cholangitis.&#x0D; Materials and methods. Depending on the magnitude of pathological changes in the biliary system, we have identified three groups of patients in whom cholangitis occurs in conditions that progress and worsen. Group I – cholangitis in conditions of choledocholithiasis, group II – cholangitis in conditions of iatrogenic strictures of the main bile ducts, and group III – cholangitis in conditions of already performed reconstruction of the duct system. The functional activity of phagocytic cells was studied.&#x0D; Results and their discussion. It was established that the functional capabilities of neutrophils are depleted in proportion to the severity of cholangitis and its duration. In case of chronic undulating cholangitis, there is an insufficiency of the phagocytic link, phagocytes work on the verge of exhausting their reserve capabilities. Adhesion forces and the potential of intracellular lysosomal enzymes are especially affected.&#x0D; Conclusions. The functional capacity of phagocytic cells in cholangitis is characterized by a deficiency, the degree of virulence is to lie down in the severity of inflammation and thrive. The greatest manifestations of changes were revealed in acid-independent mechanisms of phagocytosis, which were characterized by impaired adhesion and the release of intracellular lysosomal enzymes.

Functional Characterization of Largemouth Bass (Micropterus salmoides) Soluble FcγR Homolog in Response to Bacterial Infection.

Fc receptors (FcRs) are key players in antibody-dependent cellular phagocytosis (ADCP) with their specific recognition of the Fc portion of an immunoglobulin. Despite reports of FcγR-mediated phagocytosis in mammals, little is known about the effects of soluble FcγRs on the immune response. In this study, FcγRIα was cloned from the largemouth bass (Micropterus salmoides) (MsFcγRIα). Without a transmembrane segment or a cytoplasmic tail, MsFcγRIα was identified as a soluble form protein and widely distributed in the spleen, head kidney, and intestine. The native MsFcγRIα was detected in the serum of Nocardia seriolae-infected largemouth bass and the supernatants of transfected HEK293 cells. Additionally, it was verified that the transfected cells' surface secreted MsFcRIα could bind to largemouth bass IgM. Moreover, the expression changes of MsFcγRIα, Syk, and Lyn indicated that MsFcγRIα was engaged in the acute phase response to bacteria, and the FcγR-mediated phagocytosis pathway was activated by Nocardia seriolae stimulation. Furthermore, recombinant MsFcγRIα could enhance both reactive oxygen species (ROS) and phagocytosis to Nocardia seriolae of leukocytes, presumably through the interaction of MsFcγRIα with a complement receptor. In conclusion, these findings provided a better understanding of the function of soluble FcγRs in the immune response and further shed light on the mechanism of phagocytosis in teleosts.

Fcγ receptor-mediated phagocytosis pathway was involved in phagocytosis of mIgM+ B lymphocytes from largemouth bass (Micropterus salmoides).

The potential for phagocytosis has been proven in teleost B cells, but the research on the regulatory mechanism of phagocytosis remains lacking. In this study, three largemouth bass (Micropterus salmoides) (15 ± 5g) were injected intraperitoneally with Nocardia seriolae (105 CFU/100μl/fish) in vivo, and their spleen was collected at 72 h post-infection for mRNA-seq. After the de novo assembly of the paired-end reads, 73,622 unigenes were obtained. Gene expression profiling revealed that 2043 unigenes were differentially expressed after N. seriolae infection, comprising 1285 upregulated and 758 downregulated unigenes (q-value <0.05, log2FC>|2|) of which 181 genes were involved in phagocytosis. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis demonstrated that 12 differentially expressed genes (DEG) associated with phagocytosis were enriched in the Fcγ receptor-mediated phagocytosis signalling pathway. In vitro, the phagocytic ability of mIgM+ B lymphocytes was validated using indirect immunofluorescence assay (IIFA) and fluorescence activating cell sorter (FACS), and the phagocytosis rates of the mIgM+ B lymphocytes incubated with a Lyn inhibitor had decreased from 18.533 ± 6.00% to 11.610 ± 4.236% compared with the unblocked group. These results suggested that the Fcγ receptor-mediated phagocytosis signalling pathway had participated in the phagocytosis of B cells and provide further insight into the role of B cells in innate immunology.

Wasteosomes (corpora amylacea) of human brain can be phagocytosed and digested by macrophages

BackgroundCorpora amylacea of human brain, recently renamed as wasteosomes, are granular structures that appear during aging and also accumulate in specific areas of the brain in neurodegenerative conditions. Acting as waste containers, wasteosomes are formed by polyglucosan aggregates that entrap and isolate toxic and waste substances of different origins. They are expelled from the brain to the cerebrospinal fluid (CSF), and can be phagocytosed by macrophages. In the present study, we analyze the phagocytosis of wasteosomes and the mechanisms involved in this process. Accordingly, we purified wasteosomes from post-mortem extracted human CSF and incubated them with THP-1 macrophages. Immunofluorescence staining and time-lapse recording techniques were performed to evaluate the phagocytosis. We also immunostained human hippocampal sections to study possible interactions between wasteosomes and macrophages at central nervous system interfaces.ResultsWe observed that the wasteosomes obtained from post-mortem extracted CSF are opsonized by MBL and the C3b complement protein. Moreover, we observed that CD206 and CD35 receptors may be involved in the phagocytosis of these wasteosomes by THP-1 macrophages. Once phagocytosed, wasteosomes become degraded and some of the resulting fractions can be exposed on the surface of macrophages and interchanged between different macrophages. However, brain tissue studies show that, in physiological conditions, CD206 but not CD35 receptors may be involved in the phagocytosis of wasteosomes.ConclusionsThe present study indicates that macrophages have the machinery required to process and degrade wasteosomes, and that macrophages can interact in different ways with wasteosomes. In physiological conditions, the main mechanism involve CD206 receptors and M2 macrophages, which trigger the phagocytosis of wasteosomes without inducing inflammatory responses, thus avoiding tissue damage. However, altered wasteosomes like those obtained from post-mortem extracted CSF, which may exhibit waste elements, become opsonized by MBL and C3b, and so CD35 receptors constitute another possible mechanism of phagocytosis, leading in this case to inflammatory responses.

Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis

The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.

Brucella Phagocytosis Mediated by Pathogen-Host Interactions and Their Intracellular Survival.

The Brucella species is the causative agent of brucellosis in humans and animals. So far, brucellosis has caused considerable economic losses and serious public health threats. Furthermore, Brucella is classified as a category B bioterrorism agent. Although the mortality of brucellosis is low, the pathogens are persistent in mammalian hosts and result in chronic infection. Brucella is a facultative intracellular bacterium; hence, it has to invade different professional and non-professional phagocytes through the host phagocytosis mechanism to establish its lifecycle. The phagocytosis of Brucella into the host cells undergoes several phases including Brucella detection, formation of Brucella-containing vacuoles, and Brucella survival via intracellular growth or being killed by host-specific bactericidal activities. Different host surface receptors contribute effectively to recognize Brucella including non-opsonic receptors (toll-like receptors and scavenger receptor A) or opsonic receptors (Fc receptors and complement system receptors). Brucella lacks classical virulence factors such as exotoxin, spores, cytolysins, exoenzymes, virulence plasmid, and capsules. However, once internalized, Brucella expresses various virulence factors to avoid phagolysosome fusion, bypass harsh environments, and establish a replicative niche. This review provides general and updated information regarding Brucella phagocytosis mediated by pathogen-host interactions and their intracellular survival in host cells.

Antibody-mediated depletion of programmed death 1-positive (PD-1+) cells.

PD-1 immune checkpoint has been intensively investigated in pathogenesis and treatments for cancer and autoimmune diseases. Cells that express PD-1 (PD-1+ cells) draw ever-increasing attention in cancer and autoimmune disease research although the role of PD-1+ cells in the progression and treatments of these diseases remains largely ambiguous. One definite approach to elucidate their roles is to deplete these cells in disease settings and examine how the depletion impacts disease progression and treatments. To execute the depletion, we designed and generated the first depleting antibody (D-αPD-1) that specifically ablates PD-1+ cells. D-αPD-1 has the same variable domains as an anti-mouse PD-1 blocking antibody (RMP1-14). The constant domains of D-αPD-1 were derived from mouse IgG2a heavy and κ-light chain, respectively. D-αPD-1 was verified to bind with mouse PD-1 as well as mouse FcγRIV, an immuno-activating Fc receptor. The cell depletion effect of D-αPD-1 was confirmed in vivo using a PD-1+ cell transferring model. Since transferred PD-1+ cells, EL4 cells, are tumorigenic and EL4 tumors are lethal to host mice, the depleting effect of D-αPD-1 was also manifested by an absolute survival among the antibody-treated mice while groups receiving control treatments had median survival time of merely approximately 30days. Furthermore, we found that D-αPD-1 leads to elimination of PD-1+ cells through antibody-dependent cell-mediate phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) mechanisms. These results, altogether, confirmed the specificity and effectiveness of D-αPD-1. The results also highlighted that D-αPD-1 is a robust tool to study PD-1+ cells in cancer and autoimmune diseases and a potential therapeutic for these diseases.

Anti-inflammatory and Immunostimulant Therapy with Lactobacillus fermentum and Lactobacillus plantarum in COVID-19: A Literature Review

Inflammatory diseases are diseases characterized by inflammatory symptoms. Acute inflammatory disease can cause dysregulation of the inflammatory immune response, thereby inhibiting the development of protective immunity against infection. Among the acute inflammatory disease is COVID-19. The initial viral infection causes the antigen-presenting cells to detect the virus through a phagocytosis mechanism in the form of macrophage and dendritic cells. Lactobacillus fermentum and L. plantarum are gram-positive bacteria potentially serving as immunomodulators caused by inflammation and immune system response. Short-chain fatty acids (SCFA) produced by Lactobacillus can induce immune response through tolerogenic dendritic cells. This probiotic bacterium can induce the production of different cytokines or chemokines. Following the results of in vitro and in vivo tests, L. fermentum and L. plantarum can induce IL-10 release to activate regulatory T-cell and inhibit tumor necrosis factor-α (TNF-α) binding activity of nuclear factor kappa B (NF-κB). Literature review showed that dysregulation of inflammatory immune response disorders due to inflammatory disease could be treated using probiotic bacteria L. fermentum and L. plantarum. Therefore, it is necessary to conduct further studies on the potential of indigenous Indonesian strains of these two bacteria as anti-inflammatory and immunostimulants.

Role of Eryptosis in Hemorrhagic Stroke.

Erythrocytes undergo certain morphological changes resembling apoptosis during senescence or in an abnormal state/site, which is termed eryptosis. This process is characterized by phosphatidylserine (PS) exposure, membrane blebbing, and cell shrinkage. Eryptotic erythrocytes are subsequently removed via macrophage-mediated efferocytosis. In hemorrhagic stroke (HS), blood within an artery rapidly bleeds into the brain tissue or the subarachnoid space, resulting in severe neurological deficits. A hypoxic, over-oxidative, and pro-inflammatory microenvironment in the hematoma leads to oxidative stress, hyperosmotic shock, energy depletion, and Cl– removal in erythrocytes, which eventually triggers eryptosis. In addition, eryptosis following intracerebral hemorrhage favors hematoma clearance, which sheds light on a common mechanism of intrinsic phagocytosis. In this review, we summarized the canonical mechanisms of eryptosis and discussed its pathological conditions associated with HS. Understanding the role of eryptosis in HS may uncover additional potential interventions for further translational clinical research.

Investigation of estrogen-like effects of parabens on human neutrophils

This study investigated the estrogen-like effects and mechanism of action most commonly used parabens: methyl- (MeP), ethyl- (EtP), propyl- (PrP) and butylparaben (BuP) in human neutrophils.Neutrophils were isolated from 50 blood donors, pre-incubated with antagonists of estrogen receptor α (ERα), ERβ and G-protein coupled estrogen receptor 1 (GPER), then incubated with MeP, EtP, PrP, BuP and 17β-estradiol (E2; 10 nM). Cytotoxic effect was evaluated by MTT test. Neutrophils apoptosis, necrosis and NETs formation were assessed in flow cytometry and confocal microscopy. The ability of the neutrophils for chemotaxis, phagocytosis, NADPH oxidase activity and generation of superoxide anion was assessed in Boyden's chamber, Park's method with latex, the NBT test, and reduction of cytochrome C, respectively. The total nitric oxide concentration was measured in neutrophils supernatants by the Griess reaction. The expression of cathepsin G, neutrophil elastase, proteinase 3, ERα, ERβ and GPER was assessed in Western blot method.In our research, parabens did not cause a cytotoxic effect on human neutrophils nor affect their lifespan. Parabens exposure did not change neutrophils functions (chemotaxis, phagocytosis, NETs formation and oxygen-dependent killing mechanism) and expression of estrogen receptors.Our results suggest that parabens do not cause estrogen receptor-mediated neutrophils-related effects at concentrations measured in the plasma of individuals using products preserved with parabens.

The Prognostic Signature and Therapeutic Value of Phagocytic Regulatory Factors in Prostate Adenocarcinoma (PRAD).

There is growing evidence that phagocytosis regulatory factors (PRFs) play important roles in tumor progression, and therefore, identifying and characterizing these factors is crucial for understanding the mechanisms of cellular phagocytosis in tumorigenesis. Our research aimed to comprehensively characterize PRFs in prostate adenocarcinoma (PRAD) and to screen and determine important PRFs in PRAD which may help to inform tumor prognostic and therapeutic signatures based on these key PRFs. Here, we first systematically described the expression of PRFs in PRAD and evaluated their expression patterns and their prognostic value. We then analyzed prognostic phagocytic factors by Cox and Lasso analysis and constructed a phagocytic factor-mediated risk score. We then divided the samples into two groups with significant differences in overall survival (OS) based on the risk score. Then, we performed correlation analysis between the risk score and clinical features, immune infiltration levels, immune characteristics, immune checkpoint expression, IC50 of several classical sensitive drugs, and immunotherapy efficacy. Finally, the Human Protein Atlas (HPA) database was used to determine the protein expression of 18 PRF characteristic genes. The aforementioned results confirmed that multilayer alterations of PRFs were associated with the prognosis of patients with PRAD and the degree of macrophage infiltration. These findings may provide us with potential new therapies for PRAD.

Src kinase mediates coelomocytes phagocytosis via interacting with focal adhesion kinase in Vibrio splendidus challenged Apostichopus japonicus

Immune cells have many efficient ways to participate in the host immunity, including phagocytosis, which is an important pathway to eliminate pathogens. Only β-integrin-mediated phagocytosis pathways have been confirmed in Apostichopus japonicus. The Src family kinases (SFKs), a class of non-receptor tyrosine kinases plays an important role in the regulation of phagocytic signals in invertebrates. However, the SFK-mediated phagocytic mechanism is largely unknown in A. japonicus. In this study, a novel SFK homologue (AjSrc) with a conservative SH3 domain, an SH2 domain, and a tyrosine kinase domain was identified from A. japonicus. Both gene and protein expression of AjSrc and phosphorylation levels increased under Vibrio splendidus challenged, reaching the highest level at 24 h. Knock-down of AjSrc could depress coelomocytes' phagocytosis by 25% compared to the control group. To better understand the mechanism of AjSrc-mediated phagocytosis, focal adhesion kinase (FAK) was identified by a Co-immunoprecipitation experiment to be verified as an interactive protein of AjSrc. The phagocytosis rates of coelomocytes were decreased by 33% and 37% in AjFAK and AjSrc + AjFAK interference groups compared with the control group, respectively. Furthermore, the phosphorylation level of AjFAK was increased and reached the maximum level at 24 h post V. splendidus infection, as the same as that of AjSrc. Our results suggested that AjSrc could mediate V. splendidus-induced coelomocytes' phagocytosis via interacting with AjFAK and co-phosphorylation. This study enriched the mechanism of phagocytosis in echinoderm and provided the new theoretical foundation for disease control of sea cucumber.

Vertical Orientation Probability Matters for Enhancing Nanoparticle-Macrophage Interaction and Efficient Phagocytosis.

The geometry of nanoparticles has a profound effect on their interactions with macrophages. For an elongated geometry, the well-known curvature-dependent phagocytosis mechanism is still under debate, presumably because another important parameter, the probability of orientation, is overlooked. To verify this hypothesis, it is demonstrated that increasing the probability of the preferred vertical orientation is an efficient strategy to significantly enhance macrophage phagocytosis and uptake. This is achieved via a well-designed hexapod nanoparticle in comparison with a monopod counterpart. The hexapod nanoparticle can achieve ≈100% close-to-vertical orientation, thereby favoring phagocytosis. This discovery provides a new insight into the design of nanomaterials for macrophage-oriented bioapplications.

ОСОБЕННОСТИ МЕХАНИЗМА ФАГОЦИТОЗА ГРИБОВ РОДА CANDIDA, ASPERGILLUS И MUCOR IN VITRO И IN VIVO

The aim of this work was to study the dynamics of phagocytosis of microscopic fungi C. albicans, A. fumigatus, and M. racemosus. The studies were carried out according to the generally accepted method using cultures of pathogens of visceral mycoses (C. albicans st. No. 253, A. fumigatus st. No. 6 and M. racemosus st. No. 195). In the heparinized blood of adult rabbits and calves 5-7 days old, 1-2 drops of a suspension of fungi were added at a concentration of 1x109 spores/cm3, excluding mycelium (pseudomycelium) per 1.0 ml of blood. The tubes were placed in a thermostat with a temperature of 370C. Blood smears were stained according to Romanovsky-Giemsa and examined under a microscope at different time intervals (2-5-10-20-30-60 min). Separate fragments of Aspergillus phagocytosis were studied in a native preparation (hanging drop). For this, a leukocyte mixture was obtained from the blood with the addition of Turk's liquid. It has been established that the presence of neutrophilic leukocytes and macrophages, which phagocytized only small fragments of fungi (conidia, blastospores), is important in the mechanism of phagocytosis of microscopic fungi C. albicans, A. fumigatus and M. racemosus. The hyphae of these fungi destroyed Langhans cells. Studies of immunocellular reactions in calves with visceral mycoses have shown that the destruction of fungi that have penetrated into their body proceeds with a certain pattern. Initially, fungal hyphae are covered with platelets, the function of phagocytes is taken over by neutrophilic leukocytes and macrophages, which “swallow” small fragments of fungi. They also try to phagocytize the hyphae of fungi, but due to their large size they fail to do this, and they are rejected. Then, lymphoid cells are involved in the process of phagocytosis, which lyse fungal hyphae with the help of enzymes (proteolytic, etc.). At the same time, the fungus is destroyed into fragments of different sizes, of which small ones are phagocytosed by neutrophilic leukocytes and macrophages, and large ones are phagocytosed by Langhans giant cells. It has been established that the generally accepted concept of "phagocytosis" is not entirely acceptable to fungi that cause visceral mycoses, since aspergillus, mucor and candida are destroyed not only and not so much by the phagocytic cells themselves, but are mainly lysed due to enzymes secreted by lymphocytes.

Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart.

[Figure: see text].

Ly6G deficiency alters the dynamics of neutrophil recruitment and pathogen capture during Leishmania major skin infection

Neutrophils represent one of the first immune cell types recruited to sites of infection, where they can control pathogens by phagocytosis and cytotoxic mechanisms. Intracellular pathogens such as Leishmania major can hijack neutrophils to establish an efficient infection. However the dynamic interactions of neutrophils with the pathogen and other cells at the site of the infection are incompletely understood. Here, we have investigated the role of Ly6G, a homolog of the human CD177 protein, which has been shown to interact with cell adhesion molecules, and serves as a bona fide marker for neutrophils in mice. We show that Ly6G deficiency decreases the initial infection rate of neutrophils recruited to the site of infection. Although the uptake of L. major by subsequently recruited monocytes was tightly linked with the concomitant uptake of neutrophil material, this process was not altered by Ly6G deficiency of the neutrophils. Instead, we observed by intravital 2-photon microscopy that Ly6G-deficient neutrophils entered the site of infection with delayed initial recruitment kinetics. Thus, we conclude that by promoting neutrophils’ ability to efficiently enter the site of infection, Ly6G contributes to the early engagement of intracellular pathogens by the immune system.

Intestinal Metabolites Influence Macrophage Phagocytosis and Clearance of Bacterial Infection

The metabolite-rich environment that is the intestinal lumen contains metabolic by-products deriving from microbial fermentation and host cell metabolism, with resident macrophages being constantly exposed to this metabolic flux. Succinate, lactate and itaconate are three metabolites secreted by primed macrophages due to a fragmented tri-carboxylic acid (TCA) cycle. Additionally, succinate and lactate are known by-products of microbial fermentation. How these metabolites impact biological functioning of resident macrophages particularly in response to bacterial infection remains poorly understood. We have investigated the potential influence of these metabolites on macrophage phagocytosis and clearance of Escherichia coli (E. coli) infection. Treatment of murine bone-marrow-derived macrophages (BMDMs) with succinate reduced numbers of intracellular E. coli early during infection, while lactate-treated BMDMs displayed no difference throughout the course of infection. Treatment of BMDMs with itaconate lead to higher levels of intracellular E. coli early in the infection with bacterial burden subsequently reduced at later time-points compared to untreated macrophages, indicative of enhanced engulfment and killing capabilities of macrophages in response to itaconate. Expression of engulfment mediators MARCKS, RhoB, and CDC42 were reduced or unchanged following succinate or lactate treatment and increased in itaconate-treated macrophages following E. coli infection. Nitric oxide (NO) levels varied while pro- and anti-inflammatory cytokines differed in secretory levels in all metabolite-treated macrophages post-infection with E. coli or in response to lipopolysaccharide (LPS) stimulation. Finally, the basal phenotypic profile of metabolite-treated macrophages was altered according to marker gene expression, describing how fluid macrophage phenotype can be in response to the microenvironment. Collectively, our data suggests that microbe- and host-derived metabolites can drive distinct macrophage functional phenotypes in response to infection, whereby succinate and itaconate regulate phagocytosis and bactericidal mechanisms, limiting the intracellular bacterial niche and impeding the pathogenesis of infection.

A genome-wide CRISPR/Cas9 screen to identify phagocytosis modulators in monocytic THP-1 cells

Phagocytosis of microbial pathogens, dying or dead cells, and cell debris is essential to maintain tissue homeostasis. Impairment of these processes is associated with autoimmunity, developmental defects and toxic protein accumulation. However, the underlying molecular mechanisms of phagocytosis remain incompletely understood. Here, we performed a genome-wide CRISPR knockout screen to systematically identify regulators involved in phagocytosis of Staphylococcus (S.) aureus by human monocytic THP-1 cells. The screen identified 75 hits including known regulators of phagocytosis, e.g. members of the actin cytoskeleton regulation Arp2/3 and WAVE complexes, as well as genes previously not associated with phagocytosis. These novel genes are involved in translational control (EIF5A and DHPS) and the UDP glycosylation pathway (SLC35A2, SLC35A3, UGCG and UXS1) and were further validated by single gene knockout experiments. Whereas the knockout of EIF5A and DHPS impaired phagocytosis, knocking out SLC35A2, SLC35A3, UGCG and UXS1 resulted in increased phagocytosis. In addition to S. aureus phagocytosis, the above described genes also modulate phagocytosis of Escherichia coli and yeast-derived zymosan A. In summary, we identified both known and unknown genetic regulators of phagocytosis, the latter providing a valuable resource for future studies dissecting the underlying molecular and cellular mechanisms and their role in human disease.

Intracellular oxidative stress induced by calcium influx initiates the activation of phagocytosis in keratinocytes accumulating at S-phase of the cell cycle after UVB irradiation

BackgroundPhagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood. ObjectiveTo clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes. MethodsThe phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM. ResultsThe phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM. ConclusionTaken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.