Putting the brakes on phagocytosis: “don't‐eat‐me” signaling in physiology and disease

Review27 May 2021free access Putting the brakes on phagocytosis: “don't-eat-me” signaling in physiology and disease Shannon M Kelley Shannon M Kelley orcid.org/0000-0002-0381-2050 Center for Cell Clearance, University of Virginia, Charlottesville, VA, USA Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Kodi S Ravichandran Corresponding Author Kodi S Ravichandran [email protected] orcid.org/0000-0001-9049-1410 Center for Cell Clearance, University of Virginia, Charlottesville, VA, USA Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA VIB-UGent Center for Inflammation Research, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Shannon M Kelley Shannon M Kelley orcid.org/0000-0002-0381-2050 Center for Cell Clearance, University of Virginia, Charlottesville, VA, USA Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA Search for more papers by this author Kodi S Ravichandran Corresponding Author Kodi S Ravichandran [email protected] orcid.org/0000-0001-9049-1410 Center for Cell Clearance, University of Virginia, Charlottesville, VA, USA Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA VIB-UGent Center for Inflammation Research, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Author Information Shannon M Kelley1,2 and Kodi S Ravichandran *,1,2,3 1Center for Cell Clearance, University of Virginia, Charlottesville, VA, USA 2Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA 3VIB-UGent Center for Inflammation Research, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium *Corresponding author. Tel: +1 434 243 6093; E-mail: [email protected] EMBO Reports (2021)22:e52564https://doi.org/10.15252/embr.202152564 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Timely removal of dying or pathogenic cells by phagocytes is essential to maintaining host homeostasis. Phagocytes execute the clearance process with high fidelity while sparing healthy neighboring cells, and this process is at least partially regulated by the balance of “eat-me” and “don't-eat-me” signals expressed on the surface of host cells. Upon contact, eat-me signals activate “pro-phagocytic” receptors expressed on the phagocyte membrane and signal to promote phagocytosis. Conversely, don't-eat-me signals engage “anti-phagocytic” receptors to suppress phagocytosis. We review the current knowledge of don't-eat-me signaling in normal physiology and disease contexts where aberrant don't-eat-me signaling contributes to pathology. Glossary 18F-FDG fluorodeoxyglucose 18F Aβ amyloid beta AD Alzheimer's disease ADCP antibody-dependent cellular phagocytosis AIHA autoimmune hemolytic anemia B2M beta-2 microglobulin C3 complement component 3 cDC2 classical dendritic cell 2 dLGN dorsal lateral geniculate nucleus F4/80 EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1) FAK focal adhesion kinase Fc fragment crystallizable Gas6 growth arrest-specific protein 6 Grb2 growth factor receptor-bound protein 2 ICAM-3 intracellular adhesion molecule-3 IFNγ interferon gamma Ig immunoglobulin IL-10 interleukin 10 IL-6 interleukin 6 ITIM immunoreceptor tyrosine-based inhibitory motif ITSM immunoreceptor tyrosine-based switch motif LILRB1 leukocyte immunoglobulin-like receptor B1 LRP1 low-density lipoprotein receptor-related protein 1 mAb monoclonal antibody MerTK mer proto-oncogene tyrosine kinase me v viable motheaten MFG-E8 milk fat globule EGF factor VIII MHC class I major histocompatibility complex class I MI myocardial infarction MIAT myocardial infarction-associated transcript MyD88 myeloid differentiation primary response 88 NF-κB nuclear factor-kappa B NK natural Killer NOD non-obese diabetic NSG NOD scid gamma PD-1 programmed cell death protein 1 PD-L1 programmed death ligand 1 PD-L2 programmed death ligand 2 PECAM-1 platelet endothelial cell adhesion molecule-1 PET/CT positron emission tomography/computed tomography PI3K phosphoinositide 3-kinase PLCγ phospholipase C gamma PS phosphatidylserine Pyk2 proline-rich tyrosine kinase 2 RGC retinal ganglion cells RhoA Ras homolog family member A RIG-I retinoic acid-inducible gene I SFK Src family kinase SH2 Src homology 2 SH3 Src homology 3 SHIP Src homology 2 domain-containing inositol polyphosphate 5-phosphatase SHP-1 Src homology region 2 domain-containing phosphatase-1 SHP-2 Src homology region 2 domain-containing phosphatase-2 Siglec sialic acid-binding immunoglobulin-type lectin SIRPα signal regulatory protein α SMCs smooth muscle cells SP-A surfactant protein-A SP-D surfactant protein-D Syk spleen tyrosine kinase TAM tumor-associated macrophage TGFβ transforming growth factor β TIDC tumor-infiltrating dendritic cell TLR Toll-like receptor TNFα tumor necrosis factor α TSP thrombospondin Introduction Removal of unwanted or noxious cells is important for proper development, tissue integrity, and protection against pathogenic and immunogenic damage to the host (Arandjelovic & Ravichandran, 2015). Phagocytosis is a highly efficient process, and dying cells are rarely observed in vivo during homeostasis despite routine cellular turnover in several tissues (Elliott & Ravichandran, 2016). The “clearance crew” mediating phagocytosis can be divided into two broad categories: “professional phagocytes” and “non-professional phagocytes” (Arandjelovic & Ravichandran, 2015). Innate immune cells (e.g., immature dendritic cells, monocyte-derived macrophages, tissue-resident macrophages, and microglia) are considered professional phagocytes as they can sense and migrate toward target cells, as well as rapidly and successively internalize multiple targets (Elliott et al, 2009; Ariel & Ravichandran, 2016; Medina et al, 2020). Other tissue-resident cells (e.g., epithelial cells, fibroblasts, and endothelial cells) also play important roles in phagocytosis, albeit less efficiently, and are considered non-professional phagocytes (Wood et al, 2000; Monks et al, 2005; Juncadella et al, 2013; Arandjelovic & Ravichandran, 2015; Lee et al, 2016; Davies et al, 2018). The phagocytic response can be described in four phases: “smell”, “taste”, “ingestion”, and “digestion/response”; and the molecular details promoting the clearance of dying cells have been excellently reviewed (Arandjelovic & Ravichandran, 2015; Morioka et al, 2019). The phagocytic process must be tightly controlled, however, to prevent unwarranted removal of healthy cells. Regulation during the taste phase involves “integrated” recognition of eat-me signals and don't-eat-me signals expressed on the target cell. Eat-me signals include antibody and complement opsonins, exposed phosphatidylserine (PS), calreticulin, oxidized low-density lipoprotein, cell-bound thrombospondin (TSP), modified intracellular adhesion molecule ICAM-3, annexin I, and other modifications to surface proteins (Arandjelovic & Ravichandran, 2015). While living cells generally do not express eat-me signals, there are specific physiologic contexts when some living cells transiently express markers that partially mimic a dying cell such as during lymphocyte activation, skeletal muscle formation, and sperm–egg fusion (Elliott et al, 2005; Gardai et al, 2005; Hochreiter-Hufford et al, 2013; Hochreiter-Hufford et al, 2015; Rival et al, 2019). Thus, the expression of don't-eat-me signals provides an additional regulatory mechanism to prevent unwarranted clearance of “healthy” cells. In this review, we discuss the current knowledge of don't-eat-me signaling in phagocytosis with an emphasis on anti-phagocytic receptors that mediate the recognition of don't-eat-me signals. Most known anti-phagocytic receptors are single-pass, type I transmembrane proteins belonging to the immunoglobulin (Ig) superfamily that contain one or more immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic tail (Daeron et al, 2008). The canonical ITIM consensus sequence is (I/V/L)xYxx(L/V), where x can be any amino acid. Many anti-phagocytic receptors also have non-canonical “ITIM-like” motifs with a more divergent sequence (I/V/L/SxYxxL/V/I), as well as immunoreceptor tyrosine-based switch motifs (ITSM) and other protein-binding domains (Ravetch & Lanier, 2000; Daeron et al, 2008). In this review, we refer to all inhibitory motifs as ITIMs in an effort to reduce complexity. Anti-phagocytic receptor activation induces tyrosine phosphorylation of the cytoplasmic ITIMs and binding of cytosolic SH2 domain-containing phosphatases, such as the SHP tyrosine phosphatases and the SHIP1/2 inositol phosphatases (Neel et al, 2003; Daeron et al, 2008). These phosphatases act as downstream effectors to mediate the inhibitory function of anti-phagocytic receptors, although their downstream substrates have been challenging to identify. In antibody-dependent cellular phagocytosis (ADCP) of opsonized targets, the FcγRIIb inhibitory receptor plays an important regulatory role via activation of SHIP1/2 (Aman et al, 2000; Getahun & Cambier, 2015). Most other anti-phagocytic receptors dependent on SHP-1 and/or SHP-2 tyrosine phosphatases for their inhibitory function, and we will focus our discussion on anti-phagocytic receptor signaling mediated by these tyrosine phosphatases (Getahun & Cambier, 2015). Structurally, SHP-1 and SHP-2 contain two N-terminal SH2 domains, a catalytic phosphatase domain and a C-terminal tail (Lorenz, 2009). Phosphorylation of two tandem ITIMs is thought to be required for SHP binding and activation downstream of anti-phagocytic receptors, but the exact molecular details are not entirely clear (Lorenz, 2009; Getahun & Cambier, 2015). We will first discuss the CD47-SIRPα axis, as it represents one of the better characterized don't-eat-me checkpoints. Next, we will highlight what is known about other don't-eat-me checkpoints, and discuss several diseases with potential links to dysregulated phagocytosis as a result of aberrant anti-phagocytic signaling. SIRPα as a prototypic anti-phagocytic receptor Molecular characterization of SIRPα SIRP⍺ (also known as SIRP⍺1, PTPNS1, SHPS-1, BIT, p84, MFR, MyD-1, and CD172a) is a 115–120 kDa glycoprotein of the SIRP paired receptor family (Fujioka et al, 1996; Comu et al, 1997; Kharitonenkov et al, 1997; Yamao et al, 1997; Matozaki et al, 2009). It is expressed in most tissues and enriched on monocytes, macrophages, CD8α- classical type II dendritic cells (cDC2), neutrophils, and osteoclasts, as well as microglia and neurons. It is also moderately expressed on fibroblasts, endothelial cells, and some epithelial cells (Adams et al, 1998; Veillette et al, 1998; Johansen & Brown, 2007). The extracellular region of SIRPα contains an N-terminal IgV domain followed by two IgC domains (Fig 1) (Fujioka et al, 1996; Kharitonenkov et al, 1997; Yamao et al, 1997). Species and tissue-specific differences in the molecular weight and ligand affinity of SIRPα are attributed to the highly polymorphic IgV domain and the varying number of N-linked glycosylation sites in the extracellular region (Yamao et al, 1997; Takenaka et al, 2007). Conversely, the cytoplasmic tail of SIRPα is highly conserved with four ITIMs and a proline-rich region predicted to bind SH3 domain-containing proteins (Fig 1) (Fujioka et al, 1996; Kharitonenkov et al, 1997; Yamao et al, 1997; Veillette et al, 1998). Although originally identified in 1990 as an adhesion protein on neurons, SIRPα was independently characterized as an ITIM-containing receptor and a substrate of activated receptor tyrosine kinases in response to various growth factors and mitogens (Chuang & Lagenaur, 1990; Fujioka et al, 1996; Noguchi et al, 1996; Comu et al, 1997; Kharitonenkov et al, 1997; Yamao et al, 1997; Veillette et al, 1998). Additionally, SIRPα is tyrosine phosphorylated in response to integrin-mediated adhesion to specific extracellular matrix components (Tsuda et al, 1998; Oh et al, 1999). The tyrosine kinases responsible for direct phosphorylation of SIRPα have remained unclear and may be context-specific; however, Src family kinases (SFK), as well as focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), have been implicated in SIRPα signaling (Takeda et al, 1998; Timms et al, 1999). Nonetheless, tyrosine phosphorylation of SIRPα at specific ITIMs permits the binding of SHP-1 or SHP-2 via their SH2 domains, which relieves repression on the catalytic phosphatase domain and allows for dephosphorylation of their respective substrates (Neel et al, 2003; Lorenz, 2009). It remains unclear whether these phosphatases compete for the same binding site(s), or bind distinct sites on SIRPα; however, current evidence supports the latter model (Takada et al, 1998; Myers et al, 2020). In addition to being a binding partner, SIRPα is also a substrate of these phosphatases (Timms et al, 1998). In professional phagocytes such as macrophages, SHP-1 predominantly mediates inhibitory signaling downstream of SIRPα, consistent with the abundant expression of SHP-1 in hematopoietic cells (Veillette et al, 1998; Lorenz, 2009; Abram & Lowell, 2017). Conversely, SHP-2 is ubiquitously expressed and predominately binds SIRPα in non-hematopoietic cells in response to stimulation with growth factors, mitogens, and cellular adhesion (Kharitonenkov et al, 1997; Tsuda et al, 1998; Barclay & Van den Berg, 2014). While SHP-2 can modulate cytokine receptor signaling and macrophage activation states, whether it has a direct role in regulating phagocytosis is unclear (Neel et al, 2003; Tao et al, 2014; Niogret et al, 2019). Figure 1. Inhibition of phagocytosis by SIRP⍺ Engagement of the CD47-SIRP⍺ axis occurs in cis and in trans, which induces tyrosine phosphorylation of the ITIMs in the cytoplasmic tail of SIRP⍺. Phosphorylation at Y429 and Y453 of human SIRPα mediate binding of tyrosine phosphatase SHP-1 (Myers et al, 2020). Dephosphorylation of non-muscle myosin IIA is one proposed substrate of SHP-1, resulting in disassembly of the actomyosin cytoskeleton. (top) Viable cells express don't-eat-me signals such as CD47 and lack expression of eat-me signals, thus limiting phagocytic clearance. (bottom-left) Antibody-dependent cellular phagocytosis (ADCP) is also negatively regulated by the CD47-SIRPα axis (Zent & Elliott, 2017). (bottom-right) Efferocytosis by alveolar macrophages in the lung can be negatively regulated by SIRPα activation in response to binding CD47 and surfactant proteins, SP-A and SP-D. The mechanism of this suppression is thought to involve activation of the GTPase RhoA (Janssen et al, 2008). Download figure Download PowerPoint The best-known ligand of SIRPα is CD47 (also known as integrin-associated protein, OV-3, and Rh-related protein), which is a conserved, ubiquitously expressed 45–55-kDa transmembrane glycoprotein belonging to the Ig superfamily (Brown et al, 1990; Lindberg et al, 1993; Lindberg et al, 1994; Reinhold et al, 1995; Jiang et al, 1999). CD47 has a single N-terminal extracellular IgV domain followed by five hydrophobic membrane-spanning segments and a short C-terminal cytoplasmic tail that is alternately spliced to form four isoforms (Fig 1) (Brown et al, 1990; Lindberg et al, 1993; Reinhold et al, 1995). CD47-SIRPα binding via their IgV domains regulates many cellular processes, including leukocyte transmigration, lymphocyte homeostasis, dendritic cell maturation and activation, and bone resorption (Latour et al, 2001; de Vries et al, 2002; Liu et al, 2002; Hagnerud et al, 2006; Van et al, 2006; Lundberg et al, 2007; Saito et al, 2010; Legrand et al, 2011; Maile et al, 2011; Sato-Hashimoto et al, 2011). As we will discuss in the following sections, it is also one of the most studied don't-eat-me checkpoints in suppressing phagocytosis. CD47 is a marker of “self” for phagocytosis Seminal work by Oldenborg et al (2000) established CD47 as a marker of “self” on erythrocytes to prevent their premature clearance from circulation. The average circulating lifespan of wild-type murine erythrocytes is between 40 and 60 days (Goodman & Smith, 1961; Ishikawa-Sekigami et al, 2006; Oldenborg, 2013). In stark contrast, CD47-negative erythrocytes were shown to be rapidly cleared from circulation within 24 h of transfusion into syngeneic wild-type recipient mice in a manner independent of complement and adaptive immunity. Rather, splenic red pulp macrophages were found to facilitate the clearance of CD47-negative erythrocytes, as splenectomy and depletion of macrophages using liposomal clodronate permitted their circulation in wild-type mice. Moreover, blockade of SIRPα on wild-type splenic macrophages enhanced the clearance of wild-type erythrocytes ex vivo. Since this work, several studies over the past two decades have shown that other cell types use CD47 to avoid phagocytosis, including platelets, lymphocytes, and hematopoietic stem cells (Blazar et al, 2001; Yamao et al, 2002; Olsson et al, 2005; Ahrens et al, 2006; Ishikawa-Sekigami et al, 2006; Jaiswal et al, 2009; Catani et al, 2011; Kuriyama et al, 2012). These data are consistent with the mild thrombocytopenia and lymphopenia observed in mice globally deficient in either CD47 or SIRPα expression (Lindberg et al, 1996; Yamao et al, 2002; Ishikawa-Sekigami et al, 2006; Li et al, 2012). Recent evidence suggests that the CD47-SIRPα axis also regulates neuronal pruning by microglia during postnatal development (Lehrman et al, 2018). Retinal ganglion cells (RGCs) extend axons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus to synapse with relay neurons (Guido, 2008). RGC inputs need to be refined for proper development of eye-specific territories, and this process involves phagocytosis of RGC inputs by microglia in the dLGN (Guido, 2008; Schafer et al, 2012). CD47 was found enriched on active RGC inputs and its expression protected against microglial phagocytosis in a manner dependent on SIRPα (Lehrman et al, 2018). An observed reduction in CD47 on less active RGC inputs suggests that microglia use this marker, at least in part, to decide which inputs to remove (Lehrman et al, 2018). Together, these data support a role for the CD47-SIRP⍺ axis as a brake on the phagocytosis of “self”. Given the importance of the CD47-SIRPα axis in protecting host cells from phagocytosis, it is confounding that mice deficient in either CD47 or SIRPα expression do not have severe developmental or homeostatic abnormalities (Lindberg et al, 1996; Li et al, 2012). Moreover, CD47-deficient mice are tolerant to transfused syngeneic CD47-negative bone marrow cells, suggesting a more complex role for this signaling axis than previously thought (Blazar et al, 2001). A phagocytic “licensing” role for CD47 has been hypothesized, analogous to licensing of natural killer (NK) cells for functional cytotoxicity. Inhibitory receptors expressed on the surface of NK cells must interact with MHC class I molecules on host cells in order for NK cells to acquire proper cytotoxic function (Jonsson & Yokoyama, 2009). Elegant bone marrow reconstitution studies suggest that CD47 expression on non-hematopoietic cells is important for regulating CD47-dependent phagocytosis (Wang et al, 2007). Endogenous and donor CD47-negative leukocytes were tolerated in CD47-deficient mice that were either partially or fully reconstituted with wild-type bone marrow (Wang et al, 2007). Conversely, wild-type recipient mice reconstituted with bone marrow lacking CD47 expression rapidly eliminated donor CD47-negative cells. Senescent erythrocytes are known to be cleared by F4/80high splenic red pulp macrophages of yolk-sac and fetal liver origin; however, bone marrow-derived monocytes can contribute to the F4/80high splenic macrophage population to a certain degree (Schulz et al, 2012; Guilliams & van de Laar, 2015; Gonzalez & Castrillo, 2018). Interestingly, both wild-type mice and CD47-deficient mice reconstituted with wild-type bone marrow rapidly eliminated donor CD47-negative erythrocytes 24 weeks post-bone marrow transplantation (Wang et al, 2007). Perhaps, in this situation the donor monocyte-derived splenic macrophages contributed to the clearance of CD47-deficient erythrocytes. Alternatively, erythrocytes may lack expression of other don't-eat-me signals expressed on nucleated cells, thus making CD47-deficient erythrocytes more vulnerable to clearance. Mechanism notwithstanding, these findings suggest an important role for CD47 in phagocyte development and/or effector function, with relevance for CD47-based immunotherapies currently attempted. CD47-SIRPα axis in antibody-dependent cellular phagocytosis After identifying CD47 as a marker of “self” on erythrocytes, Oldenborg et al (2001) showed that the CD47-SIRPα axis suppresses pro-phagocytic signaling downstream of activated Fcγ receptors and complement receptors. In this study, IgG-opsonized CD47-negative erythrocytes were completely absent from circulation within 8 h post-transfusion into wild-type syngeneic recipient mice, whereas unopsonized CD47-negative erythrocytes and IgG-opsonized wild-type erythrocytes remained for > 24 h. These data are consistent with the earlier onset and more severe autoimmune hemolytic anemia (AIHA) observed in CD47-deficient non-obese diabetic (NOD) mice, which are prone to autoimmune diseases (Oldenborg et al, 2002; Wong et al, 2014). Moreover, IgG-opsonized wild-type and CD47-negative erythrocytes were also eliminated from circulation within 8 h post-transfusion into viable motheaten (mev/mev) recipient mice, which have markedly reduced SHP-1 phosphatase activity (Shultz et al, 1984; Kozlowski et al, 1993; Shultz et al, 1993; Oldenborg et al, 2001). While target cell binding and Fcγ receptor activation appear mostly unaffected by CD47-induced inhibitory signaling, the mechanism of suppression at least partially involves regulation of cytoskeletal elements important for target cell internalization (Lowry et al, 1998; May & Machesky, 2001; Diakonova et al, 2002; Kant et al, 2002; Tsai & Discher, 2008; Gomez & Descoteaux, 2018). Global tyrosine phosphorylation is reduced in phagocytes following activation of CD47-SIRPα signaling, including reduced tyrosine phosphorylation of the non-muscle myosin IIA motor protein, which was previously shown to be a direct substrate of SHP-1 following B-cell activation (Fig 1) (Baba et al, 2003; Tsai & Discher, 2008). CD47-SIRPα-mediated inhibition of inside-out integrin activation may also reduce macrophage spreading around the bound target cell (Tsai & Discher, 2008; Morrissey et al, 2020). Phagocytic receptors are suggested to be restricted in lateral movement on the plasma membrane due to interactions with the underlying cytoskeleton (Freeman et al, 2018). SIRPα has been shown to form clusters near FcγRI receptors in resting macrophages, and engagement of the CD47-SIRPα axis promotes receptor clustering, whereas unligated SIRPα is suggested to be excluded from the phagocytic cup following FcγRI stimulation with IgG or disruption of filamentous actin (Lopes et al, 2017). Thus, co-ligation of pro-phagocytic receptors (e.g., IgG activation of Fcγ receptors) and anti-phagocytic receptors (e.g., CD47-SIRPα axis) may function to fine-tune phagocytosis in some contexts, perhaps to allow for proper antigen digestion and presentation. In the absence of anti-phagocytic receptor co-ligation, such as during the clearance of apoptotic cells as we will discuss in the next section, these phagocytic “brakes” are mostly excluded from the phagocytic synapse. Studies investigating the role of the CD47-SIRPα axis in phagocytosis have largely focused on trans engagement of CD47 expressed on the target cell with SIRPα expressed on the phagocyte (Fig 1). It is important to note that many professional and non-professional phagocytes express both CD47 and SIRPα on their surface. Recent work investigating the potential for cis engagement of the CD47-SIRPα axis showed that loss of CD47 on macrophages also enhanced phagocytosis of unopsonized and IgG-opsonized erythrocytes in vitro (Hayes et al, 2020). Additionally, compared to wild-type macrophages, CD47-deficient macrophages bound more soluble CD47, presumably due to the lack of cis CD47-SIRPα interactions. Basal tyrosine phosphorylation of macrophage SIRPα was also reduced following deletion of macrophage CD47, and the loss of phosphorylation signal correlated with the loss of CD47 expression (Johansen & Brown, 2007; Hayes et al, 2020). Together, these data support a model whereby the SIRPα extracellular domains bend over to interact with the CD47 IgV domain in cis to influence the dynamics of cell clearance (Fig 1). Interestingly, other immune receptor-ligand pairs have been shown to engage in cis, suggesting that this may be a more general regulatory mechanism for immune cell function such as phagocytosis (Doucey et al, 2004). Whether the signaling strength downstream of SIRPα differs by binding CD47 in cis versus in trans remains unclear (Hayes et al, 2020). Localization and clustering of CD47-SIRPα signaling complexes near the phagocytic cup is clearly an important determining factor as to the potency of inhibitory signaling and the overall impact on phagocytosis. The conformational state of these molecules may also influence differences between cis and trans signaling downstream of SIRPα (Hayes et al, 2020). Together, these data support an inhibitory role for the CD47-SIRPα axis in ADCP via activation of SHP-1 and subsequent inhibition of contractile forces necessary for internalization of the target cell. CD47-SIRPα axis in apoptotic cell clearance Over 200–300 billion cells die in the human body every day as a part of routine cellular turnover, and most of these cells are thought to die by apoptosis (Arandjelovic & Ravichandran, 2015). The clearance of apoptotic cells, also referred to as efferocytosis, is not only immunologically silent, but also actively immunosuppressive via the secretion of anti-inflammatory mediators such as lactate, IL-10, and TGFβ (Gardai et al, 2003; Morioka et al, 2018; Perry et al, 2019). An early event following induction of apoptosis is loss of phospholipid asymmetry and exposure of phosphatidylserine (PS), a potent eat-me signal for efferocytosis, as well as membrane blebbing (Fig 1) (Fadok et al, 2001). Given the importance of the CD47-SIRPα axis in suppressing other forms of phagocytosis, its potential role in regulating efferocytosis has been explored, albeit to a lesser extent. Reduced CD47 expression is observed on some cell types (e.g., neutrophils, fibroblasts) following induction of apoptosis in vitro, as well as on senescent erythrocytes (Gardai et al, 2005; Khandelwal et al, 2007; Lv et al, 2015). However, CD47 expression changes are not evident on other cell types such as Jurkat T cells and thymocytes during early stages of apoptotic cell death. Changes in the localization of CD47 on the plasma membrane of dying cells have also been observed such that fewer molecules of CD47 engage SIRPα within the phagocytic synapse, thus reducing the binding avidity and inhibitory signaling strength downstream of SIRPα (Gardai et al, 2005; Lv et al, 2015). Further, tyrosine phosphorylation of SIRPα has been shown to be reduced in macrophages co-cultured with apoptotic cells, suggesting less involvement of the CD47-SIRPα axis in regulating efferocytosis. Interestingly, the activation of SIRPα signaling by cross-linking or recombinant CD47 has been shown to reduce efferocytosis in vitro (Gardai et al, 2005; Janssen et al, 2008; Lv et al, 2015). Thus, the CD47-SIRPα axis is capable of inhibiting efferocytosis, at least in some conditions. “Brakes” for phagocytosis such as the CD47-SIRPα axis may largely be excluded from the phagocytic synapse during efferocytosis to prevent delayed clearance of apoptotic cells, which can result in secondary necrosis and chronic inflammatory pathologies (Morioka et al, 2019). Paradoxically, a pro-phagocytic role for CD47 has also been suggested in efferocytosis. In the presence of serum, CD47-deficient apoptotic cells were shown to be engulfed less efficiently than their wild-type counterparts in vitro (Tada et al, 2003; Nilsson & Oldenborg, 2009). CD47 is known to bind the C-terminal RFYVVM domain of thrombospondins (TSP), which are platelet-derived soluble coagulation factors, and TSP-1 promotes efferocytosis by acting as a molecular bridge between apoptotic cells and specific pro-phagocytic receptors (e.g., CD36 and αvβ3 integrin) (Savill et al, 1992; Arandjelovic & Ravichandran, 2015). Moreover, erythrocyte aging studies suggest that oxidative stress induces a conformational change in CD47 that permits TSP-1 binding via its C-terminal domain, suggesting that under specific conditions CD47 may be converted to an eat-me signal (Gao et al, 1996a; Gao et al, 1996b; Burger et al, 2012). In some contexts, ligation of CD47 has also been reported to induce a form of cell death that is phenotypically similar to apoptosis, including PS exposure, but often lacks nuclear changes and is caspase-independent (Oldenborg, 2013). It is not clear to what extent, if