Phagocytes rely on a set of specific, conserved morphological changes in apoptotic cells in order to recognize that these cells are dying and to initiate their selective and swift clearance. These permissive “eat-me” signals include, but are not limited to, oxidized low-density lipoproteins, surface-bound thrombospondin, ICAM-3, calreticulin, C1q, and phosphatidylserine (PtdSer) exposed on the outer leaflet of the plasma membrane (1). Most “eat-me” signals are byproducts of the activation of intracellular cysteine-dependent aspartate-directed proteases (caspases), found downstream of both the intrinsic and extrinsic initiators of apoptosis [molecular pathways reviewed in Ref. (2, 3)]. Additional modifications to these surface alterations, such as the oxidation of PtdSer lipids containing linoleic or arachidonic acids, may serve to minimize the aberrant removal of cells that transiently expose PtdSer while activated (4, 5).

Phagocytes express numerous receptors that identify each apoptotic “eat-me” signal and instigate the rearrangement of the cytoskeleton to engulf the apoptotic corpse. In mammals, a large cohort of receptors carry out apoptotic cell sensing, with upwards of 10 cell surface receptors capable of recognizing exposed PtdSer either directly or indirectly through bridging molecules (1). The importance of apoptotic cell clearance in preventing systemic autoimmunity may provide sufficient biological impetus for maintaining redundant receptors. Nevertheless, as discussed below, phagocytic receptors that appear interchangeable based on their activating signal may in fact harbor differences in their downstream signaling components, expression profiles, regulation, and interactions with other receptors that provide the basis for diverse phagocyte responses following exposure to apoptotic cells.

It should be noted that recent advances have also distinguished additional forms of programmed cell death, including necroptosis and pyroptosis, which engage distinct signaling pathways, but yet also parallel the lytic dissolution seen in necrosis (6, 7). Loss of cell membrane integrity in each of these cases results in the release of molecules typically restricted in the nucleus or cytosol, which can then be sensed as damage-associated molecular patterns by phagocytes. For example, SAP-130, a component of the U2 small ribonucleoprotein complex, and F-actin unleashed from necrotic cells have each been established as ligands for innate immune receptors (8–11). The identification of signals that are uniquely released or expressed by cells that die through these distinct modalities of necrosis remains an area of active research.

The expression profile of phagocytic receptors varies notably among phagocyte populations, and may be further modulated by the surrounding microenvironment. The developing embryo represents one setting of persistent apoptotic cell death and efferocytosis without concomitant inflammatory signals. Extensive cellular proliferation and apoptosis is required for proper limb remodeling, organogenesis, and formation of synapses throughout the nervous system. Mice lacking even single components of the apoptotic signaling pathways exhibit severe developmental defects, often leading to perinatal lethality. For instance, Hao et al. utilized a knock-in approach to express a specific point mutant of Cytochrome C, thereby selectively eliminating its activation of Apaf-1 and other apoptotic components, while preserving its activity in the electron transport chain (12). By embryonic day (E) 14.5, these mice had severe overgrowth in multiple regions of the brain and insufficient skull development in comparison to WT counterparts, with these malformations resulting in embryonic or perinatal death for a majority of the mutant pups. Satellite glial cell (SGC) precursors have subsequently been characterized as a primary phagocyte population that clears excess apoptotic neurons, specifically in the embryonic dorsal root ganglion (DRG) (13). Isolated SGC precursors from E12.5 were found to express Megf10 and Pear1, two homologs of the CED-1 phagocytic receptor in Caenorhabditis elegans. shRNA-mediated knockdown of Megf10 and Pear1 expression in cultured SGC precursors significantly impeded their capacity to engulf dying DRG neurons in vitro (13). These findings indicate that MEGF10 and PEAR-1 may be required for proper clearance in the setting of the developing nervous system, though further in vivo studies would be necessary to confirm this model. In contrast, a distinct phagocytic receptor, MERTK, is not required in the embryo, although it is expressed in the brain at E14.5 (14). Genetic ablation of Tyro3, Axl, and Mertk (TAM TKO) in mice had no discernible effect on embryonic viability or development, with TAM TKO mice maintaining normal leukocyte development and numbers up until postnatal day 28 (15). Yet as mentioned above, these receptors are critical for efferocytosis in adult mice in a number of tissues, including the brain and thymus (16–18). A comprehensive analysis of the expression of and requirement for efferocytosis receptors in embryonic vs. adult phagocytes could further enhance our understanding of whether specific apoptotic cell sensors are dedicated to homeostatic clearance.

The TAM family of receptor tyrosine kinases, consisting of TYRO3, AXL, and MERTK, represent one group of apoptotic cell sensors that recognize the common “eat-me” signal of PtdSer, but are differentially employed for engulfment by diverse phagocyte populations (16). Systematic evaluation of the in vitro phagocytic capacity of resident and thioglycollate-induced peritoneal macrophages deficient in either Mertk, Axl, or Tyro3 underscored that all three TAM receptors can contribute to engulfment of apoptotic cells by peritoneal macrophages, although to differing extents; macrophages lacking Mertk had the most significant impairment in phagocytosis (19). Interestingly, bone marrow-derived dendritic cells (DCs) demonstrated a distinct reliance on Axl and Tyro3 for engulfment, rather than Mertk (19). Such divisions are also reflected in vivo—Axl and Tyro3 were not required for homeostatic removal of apoptotic cells in the thymus or the photoreceptor outer segments in the retina, while Mertk was essential for proper clearance (19). Recent studies have demonstrated that the receptor expression profile of phagocyte subsets is also dynamic in response to specific stimuli. Zagorska et al. characterized the differential regulation and utilization of AXL and MERTK phagocytic receptors by macrophages in either inflammatory or tolerogenic contexts (20). Treatment of bone marrow-derived macrophages (BMDMs) with Dexamethasone, an anti-inflammatory corticosteroid, elevated expression of MERTK and another PtdSer sensor Bai1, but not AXL. In contrast, a range of inflammatory signaling molecules, including lipopolysaccharide (LPS), polyinosinic:polycytidylic acid, IFNα, and IFNγ, were sufficient to enhance the expression of AXL in BMDMs, which expressed AXL only at low levels under normal culture conditions. In line with this, BMDMs lacking Mertk displayed severe defects in their baseline capacity to engulf apoptotic thymocytes, whereas Axl^−/− BMDMs only exhibited diminished uptake compared to WT BMDMs in inflammatory settings (20). This paradigm is also conserved in human phagocytes, as monocytes isolated from peripheral blood mononuclear cells upregulated MERTK expression in response to Dexamethasone or the combination of IL-10 and macrophage colony-stimulating factor (21). Such a “division of labor” is also not restricted to phagocytic receptor tyrosine kinases, as IL-4 was also shown to boost the expression of CD300f in a dose-dependent manner in macrophage subsets that typically do not express this receptor, including BMDMs cultured with IL-4 and peritoneal macrophages following in vivo administration of complexed IL-4 (22). CD300f levels in BMDMs were not sensitive to other cytokines tested, including the type II cytokine IL-13, which shares a receptor and has overlapping functions with IL-4 (23). Consequently, the sensitivity of apoptotic cell sensors to regulation by specific cytokines suggests that individual receptors may be equipped to help phagocytes respond to the presence of apoptotic cells in a given setting.

Intriguingly, additional signals in the tissue during infection may feedback to promote phagocytosis via specific phagocytic receptors, but without directly modulating receptor expression. Erdman et al. observed that human and murine macrophages displayed an increased capacity to clear either Plasmodium falciparum-infected or α-CD36-coated erythrocytes when pretreated with agonists to various Toll-like receptors (TLRs), including TLR2, TLR3, TLR4, and TLR9 (24). Uptake was dependent on CD36, a member of the class B scavenger receptor family; however, TLR stimulation did not alter surface expression of CD36 in the short timeframe of the assay (24). Thus, these results provide evidence for a level of cooperation between CD36 and various TLRs in order to potentiate engulfment. A similar synergy has also been described for uptake through integrins in the β₂ subfamily, which are important for the phagocytosis of complement-opsonized particles. Again, stimulation with TNFα, LPS, or platelet activating factor did not have an effect on α_Mβ₂ expression by J774 murine macrophages (25). Instead, exposure to each of these inflammatory stimuli mediated activation of RAP1, a Ras-like GTPase that enhanced α_Mβ₂ binding to C3bi-coated erythrocytes; expression of a dominant-negative form of RAP1 in macrophage cell lines abolished their capacity to phagocytose these target cells (25).

While the downstream signaling pathways for phagocytic receptors are not yet fully defined, differences in the molecular players may enable distinct functional outputs by the phagocyte. BAI-1, TIM-4, and the TAM family of phagocytic receptors each recognize the same signal on apoptotic cargo (PtdSer), but partly diverge in the ensuing intracellular cascades that are activated. Immunoprecipitation assays have demonstrated that BAI-1, a G protein-coupled receptor, physically forms a complex with ELMO-1 and Dock-180. Altogether, these molecules function as a guanine nucleotide exchange factor for Rac, thereby driving phagosome formation. All components of this signaling complex were required for maximal engulfment, as silencing of endogenous Elmo1 impaired the uptake of apoptotic thymocytes in Bai1-GFP transfected J774 murine macrophages (26). Other phagocytic receptors, including α_vβ₅ and MERTK, have also been shown to engage the ELMO-1/Dock-180 machinery (27, 28), with constitutively active MERTK only phosphorylating p130^CAS, an adaptor protein in this pathway, if α_vβ₅ is coexpressed.

As discussed in further detail in Section “Specification Through Integration,” activated TAM receptors also have the capacity to trigger additional signaling cascades, like the phosphorylation of PLCγ2 (29), or the induction of Socs1/3 to foster an active state of immunosuppression (30). In stark contrast, TIM-4 is thought to function solely as a tethering receptor, binding PtdSer via a metal-ion-dependent ligand binding site within its IgV domain (31) and securing apoptotic cargo on the surface of the phagocyte. In support of this, transient expression of either full-length Tim4 or various versions of Tim4 lacking the cytoplasmic tail all rendered LR73 fibroblasts more capable of engulfing apoptotic thymocytes than control fibroblasts (32). Moreover, ablation of Tim4 prevented cultured peritoneal macrophages from binding FAS ligand-treated thymocytes, and transformation of the mouse B-cell line Ba/F3 with Tim4 alone rescued the binding step of phagocytosis, but not internalization (33). Thus, TIM-4 requires pairing with other phagocytic receptors, such as MERTK, in order to mediate apoptotic cell engulfment in different phagocyte populations (33, 34). This cooperation adds a further layer of complexity to the apoptotic cell:phagocyte interface, as the activation of additional permutations of tethering and engulfing receptors could potentially generate distinctive combinations of intracellular signaling modules.

Differences in the identity of the engulfing phagocyte may also account for variation in the resulting response to apoptotic cell recognition. The clearance of dying cells is primarily carried out by professional phagocytes—tissue-resident macrophages, monocyte-derived macrophages, and DCs—that are responsible for taking up cellular debris, sensing for any molecular patterns associated with pathogens, and processing and presenting antigen to activate the adaptive immune response. Despite the maintenance of resident professional phagocyte populations within the tissues, dedicated phagocytes and non-professional phagocytes, including neighboring epithelial cells, endothelial cells, and fibroblasts, are also indispensible for clearing apoptotic cells (35, 36). Cummings et al. ascertained that within the small intestinal lamina propria alone, three subsets of resident professional phagocytes contribute to the basal clearance of apoptotic intestinal epithelial cells (IECs) that are not shed off into the intestinal lumen (37, 38). Using a mouse model in which the Villin promoter drives expression of the diphtheria toxin (DT) receptor and GFP, the authors administered low levels of DT to induce cell death in IECs without significant inflammation, and subsequently tracked dying IEC uptake. While CD11b⁺ macrophages, CD11b⁺CD103⁺ macrophages, and CD103⁺ DCs each adopted a broadly immunosuppressive transcriptional signature in response to IEC engulfment, microarray data revealed distinctions in the precise genetic program; only two genes were consistently up- or downregulated amongst all three groups of phagocytes (37). Comparable resident mononuclear phagocyte subpopulations have been classified in other tissues, such as the kidney, which at steady-state already differ in their phagocytic capacity and expression patterns of cytokines, growth factors, and chemokine receptors (39). Collectively, these findings indicate that subsets of phagocytes, despite being exposed to the same microenvironment, maintain an intrinsic capacity to respond differently to apoptotic corpse engulfment.