Phagocytic Signaling Research Articles

ATP11C is a major flippase in human erythrocytes and its defect causes congenital hemolytic anemia.

Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca(2+) concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient's senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca(2+)-activated scrambling in both control erythrocytes and the patient's erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes.

Immunogenic versus tolerogenic phagocytosis during anticancer therapy: mechanisms and clinical translation.

Phagocytosis of dying cells is a major homeostatic process that represents the final stage of cell death in a tissue context. Under basal conditions, in a diseased tissue (such as cancer) or after treatment with cytotoxic therapies (such as anticancer therapies), phagocytosis has a major role in avoiding toxic accumulation of cellular corpses. Recognition and phagocytosis of dying cancer cells dictate the eventual immunological consequences (i.e., tolerogenic, inflammatory or immunogenic) depending on a series of factors, including the type of 'eat me' signals. Homeostatic clearance of dying cancer cells (i.e., tolerogenic phagocytosis) tends to facilitate pro-tumorigenic processes and actively suppress antitumour immunity. Conversely, cancer cells killed by immunogenic anticancer therapies may stimulate non-homeostatic clearance by antigen-presenting cells and drive cancer antigen-directed immunity. On the other hand, (a general) inflammatory clearance of dying cancer cells could have pro-tumorigenic or antitumorigenic consequences depending on the context. Interestingly, the immunosuppressive consequences that accompany tolerogenic phagocytosis can be reversed through immune-checkpoint therapies. In the present review, we discuss the pivotal role of phagocytosis in regulating responses to anticancer therapy. We give particular attention to the role of phagocytosis following treatment with immunogenic or immune-checkpoint therapies, the clinical prognostic and predictive significance of phagocytic signals for cancer patients and the therapeutic strategies that can be employed for direct targeting of phagocytic determinants.

Hybrid nanoparticles improve targeting to inflammatory macrophages through phagocytic signals

Macrophages are innate immune cells with great phenotypic plasticity, which allows them to regulate an array of physiological processes such as host defense, tissue repair, and lipid/lipoprotein metabolism. In this proof-of-principle study, we report that macrophages of the M1 inflammatory phenotype can be selectively targeted by model hybrid lipid–latex (LiLa) nanoparticles bearing phagocytic signals. We demonstrate a simple and robust route to fabricate nanoparticles and then show their efficacy through imaging and drug delivery in inflammatory disease models of atherosclerosis and obesity. Self-assembled LiLa nanoparticles can be modified with a variety of hydrophobic entities such as drug cargos, signaling lipids, and imaging reporters resulting in sub-100nm nanoparticles with low polydispersities. The optimized theranostic LiLa formulation with gadolinium, fluorescein and “eat-me” phagocytic signals (Gd-FITC-LiLa) a) demonstrates high relaxivity that improves magnetic resonance imaging (MRI) sensitivity, b) encapsulates hydrophobic drugs at up to 60% by weight, and c) selectively targets inflammatory M1 macrophages concomitant with controlled release of the payload of anti-inflammatory drug. The mechanism and kinetics of the payload discharge appeared to be phospholipase A2 activity-dependent, as determined by means of intracellular Förster resonance energy transfer (FRET). In vivo, LiLa targets M1 macrophages in a mouse model of atherosclerosis, allowing noninvasive imaging of atherosclerotic plaque by MRI. In the context of obesity, LiLa particles were selectively deposited to M1 macrophages within inflamed adipose tissue, as demonstrated by single-photon intravital imaging in mice. Collectively, our results suggest that phagocytic signals can preferentially target inflammatory macrophages in experimental models of atherosclerosis and obesity, thus opening the possibility of future clinical applications that diagnose/treat these conditions. Tunable LiLa nanoparticles reported here can serve as a model theranostic platform with application in various types of imaging of the diseases such as cardiovascular disorders, obesity, and cancer where macrophages play a pathogenic role.

Chronic STING-dependent signaling in phagocytes aggravates TREX1-contingent inflammatory disease (INM2P.353)

Abstract Patients defective in the 3’ repair exonuclease 1 (Trex1) suffer from Aicardi-Goutieres Syndrome (AGS) characterized by lethal encephalitis caused by high levels of circulating type I IFN production. Trex1-/- mice similarly suffer a short life span due to inflammatory myocarditis. Our data indicates that Trex1 is responsible for eliminating DNA species that otherwise activate STING (Stimulator of Interferon Genes), a key innate immune regulator responsible for facilitating cytosolic DNA-mediated cytokine production. Loss of STING (Trex1-/- STING-/-) was observed to dramatically extend the lifespan of Trex1-/- mice due to the prevention pro-inflammatory gene induction. However, we noted that Trex1-/- macrophages did not exhibit an augmented ability to produce cytokines following exposure to STING-dependent activators, but rather appeared chronically activated, exhibiting high basal level cytokine production. The source of STING activator was found to involve intrinsic genomic DNA left over from hematopoetic cell division processes that leaked into the cytoplasm, the elimination of which usually require Trex1. Accordingly, Trex1-manifested disease could be alleviated following adoptive transfer of normal bone marrow into Trex1-/- mice. Collectively, our data indicates that STING is responsible for pro-inflammatory gene induction induced by intrinsic self-DNA in Trex1 deficient mice and provides new insight into the molecular mechanisms underlining self-DNA-mediated disease.

The effects of red ginseng saponin fraction-A (RGSF-A) on phagocytosis and intracellular signaling in Brucella abortus infected RAW 264.7 cells

This study indicated that RGSF-A caused a marked reduction in the adherence, internalization and intracellular growth of Brucella abortus in RGSF-A-treated cells. Furthermore, a decline in the intensity of F-actin fluorescence was observed in RGSF-A-treated cells compared with untreated B. abortus-infected cells. In addition, an evaluation of phagocytic signaling proteins by Western blot analysis revealed an apparent reduction of ERK and p38α phosphorylation levels in B. abortus-infected RGSF-A-treated cells compared with the control. Upon intracellular trafficking of the pathogen, a higher number of B. abortus-containing phagosomes colocalized with LAMP-1 in RGSF-A-treated cells compared with control cells. These results strongly suggest that inhibition of B. abortus uptake could be mediated by suppression in the activation of MAPKs signaling proteins phospho-ERK 1/2, and p38 levels. On the other hand, inhibition of intracellular replication results from the enhancement of phagolysosome fusion in host macrophages. This study highlights the phagocytic and intracellular modulating effect of RGSF-A and its potential as an alternative remedy to control B. abortus infection.

Glial cell interactions and glaucoma.

Purpose of reviewThe present review describes new advances in our understanding of the role of glial cells in the pathogenesis of glaucoma. It is becoming clear that retinal glia should not be studied in isolation in glaucoma because glia have dynamic and diverse interactions with a range of different cell types that could influence the disease process.Recent findingsMicroglial activity is modulated by signals from retinal ganglion cells and macroglia that influence RGC survival in various models of injury. New studies suggest that circulating monocytic populations may play a role in mediating the immune response to glaucoma. Astrocytes have been found to develop discrete localized processes that interact with a specific subset of retinal ganglion cells, possibly responding to the expression of phagocytic signals by stressed retinal ganglion cells.SummaryRetinal glia constitute a highly versatile population that interacts with various cells to maintain homeostasis and limit disease. Defining the mechanisms that underlie glial communication could enable the development of more selective therapeutic targets, with great potential clinical applications.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me’ signals: cleavage and inhibition of phagocytosis by Lp-PLA2

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic 'eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.

A therapeutic approach towards chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency (PID) characterized by the inability of phagocytes to produce reactive oxygen intermediates (ROIs) due to a defect in the NADPH oxidase complex. Recent studies have revealed that ROIs are involved in inflammatory signaling in phagocytes, illuminating the underlying mechanisms of hyper-inflammation in CGD. CGD patients frequently suffer from CGD-associated bowel inflammation, granuloma, and life-threatening infections. Based on the discovery of the regulatory function of ROIs in the immune response, therapeutic methods for excessive inflammation focusing on inflammatory cytokines are being developed for CGD. Although hematopoietic stem cell (HSC) transplantation (HSCT) is a curative therapy for CGD, successful transplants greatly depend on HSC source selection and the degree of matching of potential donors. Gene therapy trials for PID have been performed on over 120 patients with no HLA identical donor for HSCT, and have demonstrated clinical benefits. Genotoxicity in HSC gene therapy trials has expanded our knowledge on the mechanisms of vector-associated clonal expansion of gene-modified cells, which will advance gene therapy development using self-inactivating retrovirus and lentivirus vectors. We will discuss the complications of HSCT for CGD. We will then outline the status of gene therapy approaches in the treatment of CGD.

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment in C. elegans

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.

Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment inC. elegans

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.

Rotenone induces neuronal death by microglial phagocytosis of neurons

Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces microglial activation and loss of dopaminergic neurons in models of Parkinson's disease. However, the mechanisms of rotenone neurotoxicity are still poorly defined. Here, we used primary neuronal/glial cultures prepared from rat cerebella to investigate the contribution of microglia to neuronal cell death induced by low concentrations of rotenone. Rotenone at 2.5 nm induced neuronal loss over several days without increasing the numbers of necrotic or apoptotic neurons, and neuronal loss/death could be prevented by selective removal of microglia. Rotenone increased microglial proliferation and phagocytic activity, without increasing tumour necrosis factor-α release. Rotenone-induced neuronal loss/death could be prevented by inhibition of phagocytic signalling between neurons and microglia with: cyclo(Arg-Gly-Asp-d-Phe-Val) (to block the microglial vitronectin receptor); MRS2578 (to block the microglial P2Y6 receptor); or either annexin V or an antibody against phosphatidylserine (to block exposed phosphatidylserine, a well-characterized neuronal 'eat-me' signal). As inhibition of phagocytosis by five different means prevented neuronal loss without increasing neuronal death, these data indicate that rotenone neurotoxicity is at least partially mediated by microglial phagocytosis of otherwise viable neurons (phagoptosis). Thus, neuronal loss in Parkinson's disease and other neurological diseases might be prevented by blocking phagocytic signalling.

Assessment of Tumor Antigen-Loaded Solid Lipid Nanoparticles as An Efficient Delivery System for Dendritic Cell Engineering

The work attempts to overcome tumor-associated immune tolerance using a surface-modified solid lipid nanoparticle (SLNP) delivery system for dendritic cell (DC) immunotherapy. Different formulations of SLNPs (SLNPs-alone, cationic SLNPs and mannosylated SLNPs) were prepared using tumor cell lysates. Prepared nanoparticles were characterized and their ability to activate DCs to induce a tumor cell-specific response was assessed. SLNPs induced a strong phagocytic signal to DCs without any significant toxicity. Comparatively, mannosylated SLNPs evoked an optimum and effective cell-mediated immune response with no significant toxicity. Surface-modified SLNPs may play a pivotal role in designing a clinically translatable DC-based immunotherapy for gastrointestinal malignancies. This novel approach may also facilitate the treatment of residual disease, following standard therapy.

Primary phagocytosis of viable neurons by microglia activated with LPS or Aβ is dependent on calreticulin/LRP phagocytic signalling

BackgroundMicroglia are resident brain macrophages that can phagocytose dead, dying or viable neurons, which may be beneficial or detrimental in inflammatory, ischaemic and neurodegenerative brain pathologies. Cell death caused by phagocytosis of an otherwise viable cell is called ‘primary phagocytosis’ or ‘phagoptosis’. Calreticulin (CRT) exposure on the surface of cancer cells can promote their phagocytosis via LRP (low-density lipoprotein receptor-related protein) on macrophages, but it is not known whether this occurs with neurons and microglia.MethodsWe used primary cultures of cerebellar neurons, astrocytes and microglia to investigate the potential role of CRT/LRP phagocytic signalling in the phagocytosis of viable neurons by microglia stimulated with lipopolysaccharide (LPS) or nanomolar concentrations of amyloid-β peptide1-42 (Aβ). Exposure of CRT on the neuronal surface was investigated using surface biotinylation and western blotting. A phagocytosis assay was also developed using BV2 and PC12 cell lines to investigate CRT/LRP signalling in microglial phagocytosis of apoptotic cells.ResultsWe found that BV2 microglia readily phagocytosed apoptotic PC12 cells, but this was inhibited by a CRT-blocking antibody or LRP-blocking protein (receptor-associated protein: RAP). Activation of primary rat microglia with LPS or Aβ resulted in loss of co-cultured cerebellar granule neurons, and this was blocked by RAP or antibodies against CRT or against LRP, preventing all neuronal loss and death. CRT was present on the surface of viable neurons, and this exposure did not change in inflammatory conditions. CRT antibodies prevented microglia-induced neuronal loss when added to neurons, while LRP antibodies prevented neuronal loss when added to the microglia. Pre-binding of CRT to neurons promoted neuronal loss if activated microglia were added, but pre-binding of CRT to microglia or both cell types prevented microglia-induced neuronal loss.ConclusionsCRT exposure on the surface of viable or apoptotic neurons appears to be required for their phagocytosis via LRP receptors on activated microglia, but free CRT can block microglial phagocytosis of neurons by acting on microglia. Phagocytosis of CRT-exposing neurons by microglia can be a direct cause of neuronal death during inflammation, and might therefore contribute to neurodegeneration and be prevented by blocking the CRT/LRP pathway.

Cross Talk between Engulfment Receptors Stabilin-2 and Integrin αvβ5 Orchestrates Engulfment of Phosphatidylserine-Exposed Erythrocytes

Efficient cell corpse clearance is critical for health in organisms. Apoptotic cells displaying phosphatidylserine (PS) are recognized by engulfment receptors and ingested through two conserved pathways. In one pathway, engulfment receptor brain-specific angiogenesis inhibitor 1 (BAI-1) or integrin functions upstream of ELMO/DOCK180 and activate the small GTPase Rac1. In the other pathway, engulfment receptor CED-1 or stabilin-2 acts in concert with the adaptor protein GULP to activate Rac1. Stabilin-2, a PS receptor, facilitates phagocytosis of apoptotic cells and mediates the production of anti-inflammatory cytokines. Here, we propose that the stabilin-2 extracellular domain consisting of integrin-binding fasciclin 1 (FAS1) domains coordinates the activities of the two phagocytic pathways via direct interactions with integrin. Interactions between stabilin-2 and integrin were determined using biochemical assays, including coimmunoprecipitation and fluorescence resonance energy transfer (FRET). These interactions appear to have functional relevance, since knockdown of endogenous αvβ5 expression or treatment with a function-blocking αvβ5 antibody significantly decreased stabilin-2-mediated phagocytosis in the absence of soluble factors. Our data collectively suggest that the engulfment receptors of the two phagocytic pathways communicate with each other to orchestrate engulfment of damaged erythrocytes. Coordinated phagocytic signaling would be advantageous for physiological and pathological circumstances that require rapid clearance of abnormal (apoptotic or aged) cells.

The Signalling Role of the avβ5-Integrin Can Impact the Efficacy of AAV in Retinal Gene Therapy

Sub-retinal injection of the common AAV2 pseudotypes frequently results in strong transduction of the retinal pigment epithelium (RPE) as well as the retina itself. This has been of benefit to date in human clinical trials using AAV, where the disease target is in the RPE. However, many mutations predisposing to retinal disease are located in the photoreceptor cells, present in the neural retina and not the RPE; in this case the sub-retinal injection route may cause an effective “loss” of therapeutic AAV to the RPE. The αvβ5 integrin receptor is highly expressed on the apical surface of the RPE, and is essential to the daily phagocytosis of the outer segment tips of photoreceptor cells. The transduction efficiency of AAV was tested in the retinas of β5−/− mice lacking this receptor and showing defects in photoreceptor outer segment phagocytosis. Following sub-retinal injection of AAV2/5-eGFP, fluorescence was found to be stronger and more widespread in the neural retina of β5−/− mice compared to wild-types with greatly reduced fluorescence in the RPE. Increased levels of the phagocytic signalling protein MFG-E8, the ligand for the αvβ5 integrin receptor, is found to have a moderate inhibitory effect on AAV transduction of the retina. However the opposite effect is found when only the integrin-binding domain of MFG-E8, the RGD (Arginine-Glycine-Aspartic acid) domain, was increased. In this case RGD enhanced AAV-mediated retinal transduction relative to RPE transduction. These results are presented for their relevance for the design of AAV-based retinal gene therapy strategies strategies targeting retinal/photoreceptor cells.

The neuropeptides α-MSH and NPY differentially modulate phagocytosis by macrophages (120.24)

Abstract The ocular microenvironment tightly regulates immunity to prevent inflammation. These anti-inflammatory mechanisms are partly mediated by immunomodulating neuropeptides. We previously found that alpha-melanocyte stimulating hormone (α-MSH) and Neuropeptide Y (NPY) together induce activation of myeloid suppressor-like macrophages. In this study, we examined the possibility that α-MSH and NPY also modulate phagocytosis by macrophages. The monocytic cell line RAW 246.7 was treated with α-MSH or NPY at 1 ng/ml each, a concentration produced by retinal cells in culture. The treated cells were feed florescent bioparticles of Gram(-) E. Coli or Gram(+) S. Aureus with or without opsonization. Also, tested was the formation of phagolysosomes using pH sensitive florescent E. Coli or S. Aureus bioparticles. Phagocytic activity was assayed by flow cytometry. The α-MSH and NPY treated macrophages were significantly suppressed in their capacity to phagocytize unopsonized E. coli; however, suppression of S. Aureus phagocytosis was limited to NPY treated macrophages. In addition, α-MSH/NPY co-treatment suppressed both phagocytosis and phagolysosome formation in the macrophages. There was no change in phagocytic activity if the bioparticles were opsonized with antibody or complement. Therefore, as α-MSH and NPY together induce suppressor macrophages within the ocular microenvironment, they also modulate in a stimulus-dependent manner phagocytic signals within the macrophages.

Microglia Sculpt Postnatal Neural Circuits in an Activity and Complement-Dependent Manner

Microglia are the resident CNS immune cells and active surveyors of the extracellular environment. While past work has focused on the role of these cells during disease, recent imaging studies reveal dynamic interactions between microglia and synaptic elements in the healthy brain. Despite these intriguing observations, the precise function of microglia at remodeling synapses and the mechanisms that underlie microglia-synapse interactions remain elusive. In the current study, we demonstrate a role for microglia in activity-dependent synaptic pruning in the postnatal retinogeniculate system. We show that microglia engulf presynaptic inputs during peak retinogeniculate pruning and that engulfment is dependent upon neural activity and the microglia-specific phagocytic signaling pathway, complement receptor 3(CR3)/C3. Furthermore, disrupting microglia-specific CR3/C3 signaling resulted in sustained deficits in synaptic connectivity. These results define a role for microglia during postnatal development and identify underlying mechanisms by which microglia engulf and remodel developing synapses.

Eat‐me signals: Keys to molecular phagocyte biology and “Appetite” control

Hundreds of billions of cells undergo apoptosis in our body everyday and are removed by immunologically silent phagocytosis to maintain tissue homeostasis. Impairments in phagocytosis result in autoimmune and/or degenerative diseases. Eat-me signals are the key to the recognition of extracellular cargos and the initiation of the phagocytosis process by activating phagocytic receptors and signaling cascades, and are convenient targets for therapeutic modulation. Despite their importance, eat-me signals and other phagocytosis players are mostly identified on case-by-case basis with daunting challenges. This Commentary focuses on our latest knowledge of the extracellular players, highlights our approaches to systematically map unknown pathways by functional genetic and proteomic technologies, and discusses future direction to unravel the mystery of molecular phagocyte biology.

Interferon gamma induces actin polymerization, Rac1 activation and down regulates phagocytosis in human monocytic cells

IFNγ is a potent activator and IL-10 a powerful inhibitor of macrophage functions. However, neither all cellular functions are enhanced by IFNγ nor IL-10 inhibits all cellular responses. Thus, FcγRs-mediated phagocytosis in monocyte-derived macrophages (MDM) increases after IL-10 treatment, and decreases after treatment with IFNγ, although both IL-10 and IFNγ up regulate FcγRI expression. In this work we investigated the effect of IFNγ and IL-10 on phagocytic signaling by FcγRs in MDM. Treatment with IFNγ diminished phagocytosis of IgG-opsonized SRBC (IgG–SRBC) while treatment with IL-10 increased it. These opposite effects cannot be attributed to changes in FcγR expression induced by each cytokine. Early biochemical responses mediated by FcγRs were distinctly affected by cytokine treatment. Syk phosphorylation and the rise in [Ca2+]i were higher after IL-10 treatment, whereas IFNγ treatment also increased Syk phosphorylation but had no effect on the rise in [Ca2+]i. IFNγ treatment led to increased basal levels of F-actin and this effect correlated with the decrease in phagocytosis of both IgG–SRBC and non-opsonized Escherichia coli. IL-10 did not alter F-actin basal levels, and enhanced the phagocytosis of E. coli and IgG–SRBC. The level of F-actin reached after IFNγ treatment was not further increased after stimulation with IgG-SRBC or CCL5, whereas MDM treated with IL-10 showed a slightly higher response than control cells to CCL5. IFNγ increased Rac1-GTP levels. Inhibition of PI3K with LY294002 prevented IFNγ-mediated actin polymerization. Our data suggest that IFNγ induces a higher basal level of F-actin and activation of Rac1, affecting the response to stimuli that induce cytoskeleton rearrangement such as phagocytic or chemotactic stimuli.