Phagocytic Synapse Research Articles

Antibody surface mobility amplifies FcγR signaling via Arp2/3 during phagocytosis

We report herein that the anti-CD20 therapeutic antibody, rituximab, is rearranged into microclusters within the phagocytic synapse by macrophage Fcγ receptors (FcγR) during antibody-dependent cellular phagocytosis. These microclusters were observed to potently recruit Syk and to undergo rearrangements that were limited by the cytoskeleton of the target cell, with depolymerization of target-cell actin filaments leading to modest increases in phagocytic efficiency. Total internal reflection fluorescence analysis revealed that FcγR total phosphorylation, Syk phosphorylation, and Syk recruitment were enhanced when IgG-FcγR microclustering was enabled on fluid bilayers relative to immobile bilayers in a process that required Arp2/3. We conclude that on fluid surfaces, IgG-FcγR microclustering promotes signaling through Syk that is amplified by Arp2/3-driven actin rearrangements. Thus, the surface mobility of antigens bound by IgG shapes the signaling of FcγR with an unrecognized complexity beyond the zipper and trigger models of phagocytosis.

Counteracting immunotyrosine-based signaling motifs augment zebrafish leukocyte immune-type receptor-mediated phagocytic activity

Leukocyte immune-type receptors (LITRs) represent a polymorphic and polygenic family of immunoregulatory proteins originally discovered in channel catfish (Ictalurus punctatus; IpLITRs). Belonging to the immunoglobulin superfamily (IgSF), IpLITRs are generally classified as stimulatory or inhibitory types based on their utilization of various intracellular tyrosine-based signaling motifs. While research has shown that IpLITRs can activate as well as abrogate different immune cell effector responses including phagocytosis, recent identification of LITRs within the zebrafish genome (Danio rerio; DrLITRs) revealed the existence of fish LITR-types uniquely containing counteracting stimulatory and inhibitory cytoplasmic tail (CYT) region motifs (i.e., an immunoreceptor tyrosine-based activation motif; ITAM, and immunoreceptor tyrosine-based inhibitory motif; ITIM) within the same receptor. This arrangement is unusual as these motifs typically exist on separate stimulatory (i.e., ITAM-containing) or inhibitory (i.e., ITIM-containing) immunoregulatory receptors that then co-engage to fine-tune cellular signaling and effector responses. Using a flow cytometric-based phagocytosis assay, we show here that engagement of DrLITR 1.2-expressing cells with antibody coated 4.5 μm beads causes a robust ITAM-dependent phagocytic response and reveal that its tandem ITIM motif surprisingly enhances the DrLITR 1.2-induced phagocytic activity while simultaneously decreasing the receptors ability to bind the beads. Confocal microscopy studies also revealed that the ITIM-associated inhibitory signaling molecule SHP-2 is localized to the phagocytic synapse during the phagocytic response. Overall, these results provide the first functional characterization of teleost immune receptors containing a tandem ITAM and ITIM and allow for the proposal of an intracytoplasmic tail signaling model for ITIM-mediated enhancement of ITAM-dependent cellular activation.

Propulsive cell entry diverts pathogens from immune degradation by remodeling the phagocytic synapse.

Phagocytosis is a critical immune function for infection control and tissue homeostasis. During phagocytosis, pathogens are internalized and degraded in phagolysosomes. For pathogens that evade immune degradation, the prevailing view is that virulence factors are required to disrupt the biogenesis of phagolysosomes. In contrast, we present here that physical forces from motile pathogens during cell entry divert them away from the canonical degradative pathway. This altered fate begins with the force-induced remodeling of the phagocytic synapse formation. We used the parasite Toxoplasma gondii as a model because live Toxoplasma actively invades host cells using gliding motility. To differentiate the effects of physical forces from virulence factors in phagocytosis, we employed magnetic forces to induce propulsive entry of inactivated Toxoplasma into macrophages. Experiments and computer simulations show that large propulsive forces hinder productive activation of receptors by preventing their spatial segregation from phosphatases at the phagocytic synapse. Consequently, the inactivated parasites are engulfed into vacuoles that fail to mature into degradative units, similar to the live motile parasite's intracellular pathway. Using yeast cells and opsonized beads, we confirmed that this mechanism is general, not specific to the parasite used. These results reveal new aspects of immune evasion by demonstrating how physical forces during active cell entry, independent of virulence factors, enable pathogens to circumvent phagolysosomal degradation.

Basolateral Amygdala Reactive Microglia May Contribute to Synaptic Impairment and Depressive-Like Behavior in Mice with Bone Cancer Pain.

Anxiety and depression induced by cancer-related pain disturb quality of life and willingness to survive. As a component of the limbic system, the basolateral amygdala (BLA) is critical for processing negative emotions. The reactive microglial engulfment of synapses may promote depression during adolescence. However, whether microglia phagocytose synapses to mediate cancer pain-induced depression remains unclear. The present study established a bone cancer-pain model to investigate the associationbetween dendritic spine synapses and depressive-like behavior and explore the phagocytic function of microglia in the BLA. We found that tumor-bearing mice experienced postoperative pain-related depression, and their BLAs exhibited reactive microglia, as well as phagocytic synapses. The microglial inhibitor minocycline effectively mitigated depressive behavior, synaptic damage, and the phagocytic function of microglia. Our study implicates microglia-mediated synaptic loss in the BLA may act as the pathological basis of depressive-like behavior in bone cancer pain model.

Cross-Activation of Hemichannels/Gap Junctions and Immunoglobulin-Like Domains in Innate-Adaptive Immune Responses.

Hemichannels (HCs)/gap junctions (GJs) and immunoglobulin (Ig)-like domain-containing proteins (IGLDCPs) are involved in the innate–adaptive immune response independently. Despite of available evidence demonstrating the importance of HCs/GJs and IGLDCPs in initiating, implementing, and terminating the entire immune response, our understanding of their mutual interactions in immunological function remains rudimentary. IGLDCPs include immune checkpoint molecules of the immunoglobulin family expressed in T and B lymphocytes, most of which are cluster of differentiation (CD) antigens. They also constitute the principal components of the immunological synapse (IS), which is formed on the cell surface, including the phagocytic synapse, T cell synapse, B cell synapse, and astrocytes–neuronal synapse. During the three stages of the immune response, namely innate immunity, innate–adaptive immunity, and adaptive immunity, HCs/GJs and IGLDCPs are cross-activated during the entire process. The present review summarizes the current understanding of HC-released immune signaling factors that influence IGLDCPs in regulating innate–adaptive immunity. ATP-induced “eat me” signals released by HCs, as well as CD31, CD47, and CD46 “don’t eat me” signaling molecules, trigger initiation of innate immunity, which serves to regulate phagocytosis. Additionally, HC-mediated trogocytosis promotes antigen presentation and amplification. Importantly, HC-mediated CD4+ T lymphocyte activation is critical in the transition of the innate immune response to adaptive immunity. HCs also mediate non-specific transcytosis of antibodies produced by mature B lymphocytes, for instance, IgA transcytosis in ovarian cancer cells, which triggers innate immunity. Further understanding of the interplay between HCs/GJs and IGLDCPs would aid in identifying therapeutic targets that regulate the HC–Ig-like domain immune response, thereby providing a viable treatment strategy for immunological diseases. The present review delineates the clinical immunology-related applications of HC–Ig-like domain cross-activation, which would greatly benefit medical professionals and immunological researchers alike. HCs/GJs and IGLDCPs mediate phagocytosis via ATP; “eat me and don’t eat me” signals trigger innate immunity; HC-mediated trogocytosis promotes antigen presentation and amplification in innate–adaptive immunity; HCs also mediate non-specific transcytosis of antibodies produced by mature B lymphocytes in adaptive immunity.

Macrophage activation on "phagocytic synapse" arrays: Spacing of nanoclustered ligands directs TLR1/2 signaling with an intrinsic limit.

The activation of Toll-like receptor heterodimer 1/2 (TLR1/2) by microbial components plays a critical role in host immune responses against pathogens. TLR1/2 signaling is sensitive to the chemical structure of ligands, but its dependence on the spatial distribution of ligands on microbial surfaces remains unexplored. Here, we reveal the quantitative relationship between TLR1/2-triggered immune responses and the spacing of ligand clusters by designing an artificial "phagocytic synapse" nanoarray platform to mimic the cell-microbe interface. The ligand spacing dictates the proximity of receptor clusters on the cell surface and consequently the pro-inflammatory responses of macrophages. However, cell responses reach their maximum at small ligand spacings when the receptor nanoclusters become adjacent to one another. Our study demonstrates the feasibility of using spatially patterned ligands to modulate innate immunity. It shows that the receptor clusters of TLR1/2 act as a driver in integrating the spatial cues of ligands into cell-level activation events.

A Sampling of Highlights from the Literature

Mimicking STING–cGAMP binding can activate signaling (by Kissiovd via Wikimedia Commons)How to effectively induce STING activation in the tumor microenvironment has not yet been determined. Lu et al. developed microparticles that can release an encapsulated STING agonist in waves, allowing for timed release into the tumor microenvironment (TME) without having to perform multiple intratumoral injections. Treatment of tumor-bearing mice reduces tumor growth and metastases. Chin et al. identified SR-717, a non-nucleotide small-molecule STING agonist, that mimics cGAMP and can confer the active conformation of STING to promote antitumor responses by CD8+ T cells and NK cells. Pan et al. identified MSA-2, which binds and activates human and mouse STING preferentially in the acidified TME, can be given safely as a subcutaneous and oral treatment, induces clearance of tumors, and generates long-lasting antitumor immunity. These studies highlight the potential utility of new classes of STING agonists.Lu X, …, Jaklenec A. Sci Transl Med 2020 Aug 12;12:eaaz6606.Chin EN, …, Lairson LL. Science 2020 Aug 21;369:993–9.Pan B-S, …, Addona GH. Science 2020 Aug 21;369:eaba6098.scRNA-seeking for TREM2 effects (by Mohamed Hassan via PxHere)Immune-suppressive cells support tumor growth, thus identification and targeting of these cells are crucial to induce antitumor immunity. Katzenelenbogen et al. use a novel technology that integrates scRNA sequencing with intracellular protein activity (INs-seq) to establish that TREM2 expression positively correlates with arginase-1+ myeloid cells in tumors. Knockout of TREM2 decreases myeloid cells and improves T-cell function in tumors, delaying tumor growth. Molgora et al. demonstrate that TREM2 knockout or blockade delays tumor growth in a T cell–mediated manner and improves checkpoint blockade efficacy. Thus, TREM2+ tumor-infiltrating myeloid cells inhibit antitumor immunity and are targetable.Katzenelenbogen Y, …, Amit I. Cell 2020 Aug 20;182;872–85.E19.Molgora M, …, Colonna M. Cell 2020 Aug 20;182:886–900.E17.BTN3A1 can be overexpressed in cancer (from Fig. 2 of Gu et al., Front Immunol 2015)Butyrophilins (BTNs) have a structure comparable to some B7 family inhibitory members but regulate T-cell responses through unclear mechanisms. Payne et al. show that BTN3A1 is overexpressed in high-grade ovarian cancer and disrupts αβ T-cell activation by preventing segregation of the phosphatase, CD45, from the immunologic synapse. Stabilizing BTN3A1's extracellular domain with CD277 antibodies or BTN3A1 blockade reversed this, unleashing both αβ and γδ T-cell responses. Thus, targeting inhibitory BTNs could be a potential strategy to boost antitumor responses and increase efficacy of immunotherapy.Payne KK, …, Conejo-Garcia JR. Science 2020 Aug 21;369:942–9.Altering CARs to inhibit ubiquitination (image from PickPik)Despite the efficacy of chimeric antigen receptor (CAR) T-cell therapies, many cancer patients do not respond or eventually fail with poor CAR T-cell persistence. Upon antigen binding, CARs are ubiquitinated and downregulated from the surface of T cells, and subsequently degraded intracellularly. Ubiquitination-blocking mutations in CARs reverse degradation, and lead to enhanced signaling and recycling with increased CAR surface expression. These effects improve tumor control, increase the persistence of CAR T cells in mice, and promote differentiation to central memory cells. Thus, inhibiting the ubiquitination of CARs improves the efficacy and longevity of CAR T-cell therapy.Li W, …, Wang H. Immunity 2020 Aug 18;53:456–70.E6.Activating NK cells can reduce tumor spread (by NIAID via Wikimedia Commons)How tumors evade NK-cell surveillance is not yet fully elucidated. With exposure to tumor cells, NK cells acquire a resting, rather than active, phenotype and not only lose cytotoxic ability but promote metastasis. Sequencing and receptor–ligand analysis show tumor-exposed NK cells upregulate inhibitory receptors, as well as DNA methyltransferases (DNMTs), and bind tumor cells via KLRG1 or TIGIT. Blocking tumor interaction through TIGIT or KLRG1, or inhibiting DNMTs, reduces tumor cell spread.Chan IS, …, Ewald AJ. J Cell Biol 2020 Jul 9;219:e202001134.Don't eat me! (adapted from “Children Eating and Enjoying Watermelon Fruit” via Wannapik Studio)CD47 is a “don't eat me” signal used by tumor cells to avoid being phagocytosed by macrophages via CD47 ligation of SIRPA. The biochemical basis of this “don't eat me” signal remains unclear. CD47 ligation re-localizes SIRPA to the phagocytic synapse, where it interferes with macrophage cell spreading by inhibiting inside-out activation of integrin signaling. Reactivating integrins via manganese re-induces engulfment and phagocytosis of targets with or without CD47 conjugation. Thus, activating integrins may hold utility in reversing the protective impact of tumor expression of CD47.Morrissey MA, …, Vale RD. Immunity 2020 Aug 18;53:290–302.E6.

CD47-SIRPα Axis as a Biomarker and Therapeutic Target in Cancer: Current Perspectives and Future Challenges in Nonsmall Cell Lung Cancer.

CD47 is a cell surface protein in the immunoglobulin superfamily which is normally expressed at low levels in every healthy cell. It´s main physiologic function is to act as an inhibitor of phagocytosis; this occurs throughout interaction with SIRPa expressed on macrophages. Interaction between CD47 and SIRPa leads to activation of tyrosine phosphatases that inhibit myosin accumulation at the submembrane assembly site of the phagocytic synapse, resulting in phagocytosis blockade. In this way CD47 acts as a “don´t eat me signal” for healthy self-cells; accordingly, loss of CD47 leads to phagocytosis of aged or damaged cells. Taking advantage of this anti-phagocytic signal provided by CD47, many types of tumors overexpress this protein, thereby avoiding phagocytosis by macrophages and aiding in the survival of cancer cells. The aim of this review is to describe the physiologic the pathophysiologic role of CD47; summarize the available high-quality information about this molecule as a potential biomarker and/or therapeutic target in cancer; finally, we present an in-depth analysis of the available information about CD47 in association with nonsmall cell lung cancer, EGFR mutations, and tumor microenvironment.

CD47 Ligation Repositions the Inhibitory Receptor SIRPA to Suppress Integrin Activation and Phagocytosis

CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.

Activation-induced cell protrusions and vesicle secretion of human lung macrophages revealed by super-resolution microscopy

Abstract Recent studies using super-resolution microscopy have established that the organisation of immune cell receptors impacts signal integration and cellular activation. Understanding the nano-scale dynamics of surface receptors on tissue specific macrophages is especially important, as they are phenotypically diverse and possess a large repertoire of receptors. However, tissue macrophages are highly auto-fluorescent, severely limiting the utility of light microscopy. Here, we report a novel correction technique which utilises a moving median filter to remove auto-fluorescent noise from stochastic optical reconstruction microscopy datasets. Using this, we visualised lung macrophages (LMs) activated through Fc receptors by IgG-coated glass slides, representing a 2D model of the phagocytic synapse. This unexpectedly revealed the formation of protrusions, at the surface of LMs but not blood-derived macrophages. Class I MHC protein accumulated at the tips, consistent with a role for macrophage protrusions in antigen presentation. Additionally, staining for the exosome marker CD81 revealed the secretion of extracellular vesicles. Classically, cell-derived vesicles are studied after bulk isolation, which is harsh and comes with many caveats. Imaging them directly upon secretion allows analysis of their properties on a cell-by-cell basis in a near-native state. We discovered that LM vesicles appeared distinct from those secreted from blood-derived macrophages in that their average diameter was much smaller (80 nm ± 19 nm vs. 159 nm ± 78 nm). Thus, our correction method for super-resolution microscopy revealed novel cell biology – protrusion formation and vesicle secretion – triggered upon activation of human LMs.

Membrane fluctuations and acidosis regulate cooperative binding of 'marker of self' protein CD47 with the macrophage checkpoint receptor SIRPα.

Cell-cell interactions that result from membrane proteins binding weakly in trans can cause accumulations in cis that suggest cooperativity and thereby an acute sensitivity to environmental factors. The ubiquitous 'marker of self' protein CD47 binds weakly to SIRPα on macrophages, which leads to accumulation of SIRPα (also known as SHPS-1, CD172A and SIRPA) at phagocytic synapses and ultimately to inhibition of engulfment of 'self' cells - including cancer cells. We reconstituted this macrophage checkpoint with GFP-tagged CD47 on giant vesicles generated from plasma membranes and then imaged vesicles adhering to SIRPα immobilized on a surface. CD47 diffusion is impeded near the surface, and the binding-unbinding events reveal cooperative interactions as a concentration-dependent two-dimensional affinity. Membrane fluctuations out-of-plane link cooperativity to membrane flexibility with suppressed fluctuations in the vicinity of bound complexes. Slight acidity (pH 6) stiffens membranes, diminishes cooperative interactions and also reduces 'self' signaling of cancer cells in phagocytosis. Sensitivity of cell-cell interactions to microenvironmental factors - such as the acidity of tumors and other diseased or inflamed sites - can thus arise from the collective cooperative properties of flexible membranes.This article has an associated First Person interview with the first author of the paper.

Mechanosensing of Solid Tumors by Cancer-Attacking Macrophages

The physical biology of immune cell interactions with solid tumors has many facets, from tissue and cell levels down to key molecules. Macrophages are highly phagocytic, especially when derived from marrow, but whether they can also traffic into solid tumors and engulf cancer cells is questionable, given the well-known limitations of tumor-associated macrophages (TAMs). Here, a ‘self’ recognition receptor on macrophages, termed SIRPa, was inhibited to block binding at a phagocytic synapse to its membrane ligand, the ‘‘marker of self’’ CD47 found on all other cells. The modified macrophages were then systemically injected into mice with fluorescent human tumors that were also antibody targeted. Within days, the tumors regressed, and single-cell fluorescence analyses showed that the more the macrophages engulfed, the more they accumulated within regressing tumors. Human-marrow-derived macrophages engorged on the human tumors, while TAMs were minimally phagocytic, even toward CD47-knockdown tumors. Super-resolution imaging and mathematical modeling at multiple scales deepen insight into the key processes. Consistent with tumor-selective engorge-and-accumulate processes in vivo, phagocytosis in vitro inhibited macrophage migration through micropores that mimic features of dense 3D tissue. Accumulation of SIRPa-inhibited macrophages in tumors favored tumor regression for 1-2 weeks, but donor macrophages quickly differentiated toward non-phagocytic, high-SIRPa TAMs. Analyses of macrophages on soft (like marrow) or stiff (like solid tumors) collagenous gels demonstrated a stiffness-driven upregulation of SIRPa and the mechanosensitive nuclear marker lamin-A. Mechanosensitive differentiation was similarly evident in vivo and likely limited the anti-tumor effects, as confirmed by re-initiation of tumor regression by fresh injections of SIRPa-inhibited macrophages. Macrophage motility, phagocytosis, and differentiation in vivo are thus coupled and physically modulated.

The "Phagocytic Synapse" and Clearance of Apoptotic Cells.

Apoptosis and subsequent phagocytic clearance of apoptotic cells is important for embryonic development, maintenance of tissues that require regular cellular renewal and innate immunity. The timely removal of apoptotic cells prevents progression to secondary necrosis and release of cellular contents, preventing cellular stress and inflammation. In addition, altered phagocyte behavior following apoptotic cell contact and phagocytosis engages an anti-inflammatory phenotype, which impacts upon development and progression of inflammatory and immune responses. Defective apoptotic cell clearance underlies the development of various inflammatory and autoimmune diseases. There is considerable functional redundancy in the receptors that mediate apoptotic cell clearance, highlighting the importance of this process in diverse physiological processes. A single phagocyte may utilize multiple receptor pathways for the efficient capture of apoptotic cells by phagocytes (tethering) and the subsequent initiation of signaling events necessary for internalization. In this review, we will consider the surface alterations and molecular opsonization events associated with apoptosis that may represent a tunable signal that confers distinct intracellular signaling events and hence specific phagocyte responses in a context-dependent manner. Efficient molecular communication between phagocytes and apoptotic targets may require cooperative receptor utilization and the establishment of efferocytic synapse, which acts to stabilize adhesive interactions and facilitate the organization of signaling platforms that are necessary for controlling phagocyte responses.

Efferocytosis of dying cells differentially modulate immunological outcomes in tumor microenvironment.

Programmed cell death (apoptosis) is an integral part of tissue homeostasis in complex organisms, allowing for tissue turnover, repair, and renewal while simultaneously inhibiting the release of self antigens and danger signals from apoptotic cell-derived constituents that can result in immune activation, inflammation, and autoimmunity. Unlike cells in culture, the physiological fate of cells that die by apoptosis in vivo is their rapid recognition and engulfment by phagocytic cells (a process called efferocytosis). To this end, apoptotic cells express specific eat-me signals, such as externalized phosphatidylserine (PS), that are recognized in a specific context by receptors to initiate signaling pathways for engulfment. The importance of carefully regulated recognition and clearance pathways is evident in the spectrum of inflammatory and autoimmune disorders caused by defects in PS receptors and signaling molecules. However, in recent years, several additional cell death pathways have emerged, including immunogenic cell death, necroptosis, pyroptosis, and netosis that interweave different cell death pathways with distinct innate and adaptive responses from classical apoptosis that can shape long-term host immunity. In this review, we discuss the role of different cell death pathways in terms of their immune potential outcomes specifically resulting in specific cell corpse/phagocyte interactions (phagocytic synapses) that impinge on host immunity, with a main emphasis on tolerance and cancer immunotherapy.

Abstract PR15: Engineering phagocytic signaling

Abstract Phagocytosis of specific targets in clinically-relevant settings, including engulfment of tumor cells and removal of cell corpses following aggressive cancer treatment, has been hampered by an incomplete understanding of phagocytic signaling. Therefore, we developed a cellular reconstitution platform amenable to direct, real-time imaging to dissect phagocytic signaling in molecular detail. Our gain-of-function system converts inept phagocytes into efficient ones, enabling us to appreciate the signaling behaviors associated with phagocytic proficiency. We report that 1) phagocytosis receptors form mobile microclusters in response to ligation, 2) microclusters recruit cytosolic collaborators to the plasma membrane and spatially organize actin dynamics at the phagocytic synapse, and 3) the natural ligand-receptor pair can be replaced by an engineered module, indicating that phagocytic signaling can be reprogrammed towards specific targets. Thus, we have discovered that a conserved signaling system required for recognition and removal of altered self bears unexpected mechanistic similarity to adaptive immune responses directed towards pathogens and tumor cells. In addition, our ability to replace the endogenous receptor with a heterologous system indicates that phagocytosis can be programmed in a manner conceptually similar to the Chimeric Antigen Receptor used in the clinic to program cytotoxic T cells to kill tumor cells. Our ability to engineer the process suggests that methods to re-direct phagocytes towards targets of clinical importance are possible, approaches that may enable recognition and removal of tumor cells and boost the anti-tumor immune response. Citation Format: Adam Williamson, Ron Vale. Engineering phagocytic signaling [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR15.

Comparative Anatomy of Phagocytic and Immunological Synapses.

The generation of phagocytic cups and immunological synapses are crucial events of the innate and adaptive immune responses, respectively. They are triggered by distinct immune receptors and performed by different cell types. However, growing experimental evidence shows that a very close series of molecular and cellular events control these two processes. Thus, the tight and dynamic interplay between receptor signaling, actin and microtubule cytoskeleton, and targeted vesicle traffic are all critical features to build functional phagosomes and immunological synapses. Interestingly, both phagocytic cups and immunological synapses display particular spatial and temporal patterns of receptors and signaling molecules, leading to the notion of “phagocytic synapse.” Here, we discuss both types of structures, their organization, and the mechanisms by which they are generated and regulated.

Cell rigidity and shape override CD47's “self”-signaling in phagocytosis by hyperactivating myosin-II

AbstractA macrophage engulfs another cell or foreign particle in an adhesive process that often activates myosin-II, unless the macrophage also engages “marker of self” CD47 that inhibits myosin. For many cell types, adhesion-induced activation of myosin-II is maximized by adhesion to a rigid rather than a flexible substrate. Here we demonstrate that rigidity of a phagocytosed cell also hyperactivates myosin-II, which locally overwhelms self-signaling at a phagocytic synapse. Cell stiffness is one among many factors including shape that changes in erythropoiesis, in senescence and in diseases ranging from inherited anemias and malaria to cancer. Controlled stiffening of normal human red blood cells (RBCs) in different shapes does not compromise CD47's interaction with the macrophage self-recognition receptor signal regulatory protein alpha (SIRPA). Uptake of antibody-opsonized RBCs is always fastest with rigid RBC discocytes, which also show that maximal active myosin-II at the synapse can dominate self-signaling by CD47. Rigid but rounded RBC stomatocytes signal self better than rigid RBC discocytes, highlighting the effects of shape on CD47 inhibition. Physical properties of phagocytic targets thus regulate self signaling, as is relevant to erythropoiesis, to clearance of rigid RBCs after blood storage, clearance of rigid pathological cells such as thalassemic or sickle cells, and even to interactions of soft/stiff cancer cells with macrophages.

Physics vs Biology of Phagocytosis: Cell Rigidity and Shape Override CD47 ‘Self’ Signaling in Phagocytosis by Hyperactivating Myosin-II

A macrophage engulfs another cell or foreign particle in an adhesive process that often activates Myosin-II, unless the macrophage also engages ‘Marker of Self’ CD47 that inhibits Myosin. Adhesion processes of many cell types also activate Myosin and increasingly so on rigid substrates. Here we demonstrate that the rigidity of a phagocytosed RBC hyperactivates Myosin to overwhelm ‘Self’ signaling to macrophages. RBC stiffness is one among many factors, including shape, that change in senescence, some anemias, and diseases such as malaria. Controlled stiffening of normal human RBCs in different shapes did not compromise interactions of CD47 on RBCs with the macrophage ‘Self’-recognition receptor, SIRPa. Uptake of antibody-opsonized RBC was always fastest with rigid RBC-Discocytes, which also showed maximal active Myosin at the phagocytic synapse that biophysically out-competed ‘Self’ signaling by CD47. Rigid but more rounded RBC-Stomatocytes signaled ‘Self’ moreso than rigid RBC-Discocytes, highlighting the effects of shape. Physical properties of phagocytic targets can thus modulate ‘Self’ signaling as seems relevant to splenic clearance of rigid red cells after storage or clearance of rigid pathological cells such as sickle and thalassemic red cells.

Synaptic view of eukaryotic cell

Synapses are stable adhesive domains between two neighbouring cells of the multicellular organisms which serve for cell–cell communication as well as for information processing and storing. The synaptic concept was developed over more than 100 years specifically for neuronal cell–cell communication. In the last ten years, this concept was adapted to embrace other cell–cell communication phenomena. Here, we focus on the recently emerged phagocytic synapse and propose new endosymbiotic synapses and “intracellular organellar synapses”. All these synapses of eukaryotic cells are in a good position to explain the high capacity of eukaryotic cells for integration of diverse signalling inputs into coherent cellular behaviour.

The phagocytic synapse in distinguishing particulate and soluble stimuli

The phagocytic synapse in distinguishing particulate and soluble stimuli