TAM receptors in phagocytosis: Beyond the mere internalization of particles

TYRO3, AXL, and MERTK constitute the TAM family of receptor tyrosine kinases, activated by their ligands GAS6 and PROS1. TAMs are necessary for adult homeostasis in the immune, nervous, reproductive, skeletal, and vascular systems. Among additional cellular functions employed by TAMs, phagocytosis is central for tissue health. TAM receptors are dominant in providing phagocytes with the molecular machinery necessary to engulf diverse targets, including apoptotic cells, myelin debris, and portions of live cells in a phosphatidylserine‐dependent manner. Simultaneously, TAMs drive the release of anti‐inflammatory and tissue repair molecules. Disruption of the TAM‐driven phagocytic pathway has detrimental consequences, resulting in autoimmunity, male infertility, blindness, and disrupted vascular integrity, and which is thought to contribute to neurodegenerative diseases. Although structurally and functionally redundant, the TAM receptors and ligands underlie complex signaling cascades, of which several key aspects are yet to be elucidated. We discuss similarities and differences between TAMs and other phagocytic pathways, highlight future directions and how TAMs can be harnessed therapeutically to modulate phagocytosis.


| COMP ONENTS OF THE TAM S IG NALING PATHWAY
6][7][8][9] On the intracellular carboxy-terminus, the TAM receptors bear their tyrosine kinase domains which, upon phosphorylation, trigger various intracellular signaling cascades.The TAM receptors were discovered and isolated by independent groups, and subsequently underwent several changes in nomenclature. 1The identification of their complete cDNA revealed sequence and structural homology, 7,8,10 and allowed their segregation in an independent category of RTKs. 1 They remained orphan receptors for some time, until their ligands were identified in 1995. 11Although TAM ligands GAS6 and PROS1 share high structural and functional homology, there are also distinctions, including their binding affinities to the TAM receptors.2][13][14] PROS1 activates TYRO3 and MERTK, but not AXL. 15,16GAS6 and PROS1 are large proteins (~70 kDa), and share similar structure with 42% homology.As shown in Figure 1, their amino-terminus encodes for a Gla domain, followed by four EGF-like repeats.The Gla domain, present in a dozen proteins, is a ~45 amino acids long stretch, 11,17,18 rich in glutamic acid residues (either 11 or 13 glutamic acid residues, depending on the protein and species), which are post-translationally γ-carboxylated in a vitamin K-dependent manner (Figure 1). 19,20is γ-carboxylation determines TAM ligand bioactivity, 15,16 as explained in detail in the next paragraphs.The carboxy-terminus of PROS1 and GAS6 contains a "sex hormone-binding globulin" (SHBG) domain, itself composed of two laminin G domains.2][23] As receptor tyrosine kinases, the TAMs dimerize upon ligand binding, allowing for either homo-or heterodimerization.Similarly, GAS6 and PROS1 may bind their respective TAMs contributing to active signaling either as homo-or hetero-dimers.To date, the specific ligand-receptor combinations and their respective downstream signaling pathways are mostly undetermined.Being secreted ligands, they can activate TAMs both in an autocrine and paracrine manner.After ligand binding and TAM dimerization, the receptor's tyrosine intracellular domains are phosphorylated, exposing docking sites for several intracellular molecules carrying out signal transduction.The phosphoinositide 3 kinase (PI3K)/AKT pathway, the phospholipase C, ERK1/2, Ras, and MAP kinase activation are commonly activated signaling cascades in most TAM-expressing cells.Moreover, the JAK/STAT signaling pathway may often prevail over the others, determining differential TAM bioactivity and functional diversity. 9,24TAM activation culminates in the regulation of several physiological processes, including cellular proliferation, survival and growth, immune functions such as cytokines and reactive oxygen species (ROS) release, phagocytosis and efferocytosis. 1,9,25,26Figure 1 presents a visualization of TAM receptor-ligand interactions, and the intracellular pathways driving phagocytosis, also elaborated below.
Loss of function of any of the TAM receptors in murine models does not lead to any major developmental issue, even in triple Knockout (KO) mice deleted for all three TAMs. 4,27,28Postnatal triple KO mice, however, develop male sterility, retinal degeneration, autoimmune-like disorders, and neurodegeneration, 24,[27][28][29][30][31] indicating that TAM signaling is necessary to preserve homeostasis in adult tissues.GAS6 mutants are generally healthy and do not develop the postnatal phenotypes mentioned for TAM triple KO mice.Reduced GAS6 levels even confer protection against thrombosis, 32 liver disease, 33 and cancer metastasis. 34The ability of PROS1 to activate the TAM receptors has been under debate for many years. 14,35,368][39][40] PROS1 is mostly known as a potent blood anticoagulant, and, predictably, deleting Pros1 systemically causes prenatal death due to coagulopathy and impaired vasculogenesis, with severe hemorrhage. 41,42Despite the functional redundancy of TAM receptors and their ligands, how this pathway regulates extremely diverse cellular functions affecting numerous features of cellular physiology is still incompletely understood.In the following paragraphs, we will focus on the role of TAMs in phagocytosis and inflammation-two important and functionally linked determinants of adult tissue well-being.

| TAM RECEP TOR-MED IATED PHAG O C Y TOS IS: THE MECHANIS TIC S
4][45] Like in a feast, different diners recognize and prefer different foods.After making a choice and intake of the preferred food, come satiation and digestion.These humanized terms translate molecularly into recognition, contact, uptake and recycling, with specific molecules involved in the recognition (find me signals), contact (eat me signals) and downstream "digestion" activities.Many forms of phagocytosis exist, from uptake of intruders such as bacteria (by neutrophils), through the localized and limited pruning of cell fractions as in photoreceptor outer segments (by retinal pigment epithelium, RPE) and neuronal synapses (by microglia), to ingesting cellular debris (e.g., myelin fragments by microglia) or entirely devouring dead cells (by macrophages).Each form of phagocytosis is not only tailored to the physiological context but also molecularly regulated and executed by specific proteins.In this context, the TAMs were identified for removing apoptotic cells (ACs), a type of phagocytosis termed efferocytosis, 46 but are now also recognized in the localized pruning of portions from viable cells.
Efferocytosis is mechanistically very similar to phagocytosis, but functionally very different.Phagocytosis engages molecular players and intracellular signaling cascades aimed at building up the immune response, with antigen presentation as well as release of proinflammatory cytokines.On the contrary, efferocytosis is, from the immune point of view, a "silent" and counter-inflammatory form of clearance, that further signals tissue repair. 47,48This type of phagocytic engulfment is extremely important for tissues which cannot shed their dead cells to the environment, as done for skin keratinocytes or intestinal epithelium.This would be the case for clearing apoptotic neutrophils within tissues during the resolution phase of F I G U R E 1 TAM receptor-ligand structure, and signaling in phagocytosis.Both ligands and receptors present high structural homology.The TAM ligands GAS6 and PROS1 bind to phosphatidylserine, which has been externalized by apoptotic cells, via the Gla domain at their N-terminus.The Gla domain is followed by four EGF-like repeats, and a SHBG domain on their carboxy-terminus, which constitutes the binding site to the TAM receptors' Ig-domains, on their extracellular N-terminal portion.Subsequently the TAM receptors present two fibronectin type III (FNIII) repeats, the transmembrane spanning portion of the molecule, and the protein-tyrosine kinase (PTK) domains on the intracellular carboxy-terminus (Inset blow-up).The vitamin K-dependent post-translational γ-carboxylation of the Gla domain allows GAS6/PROS1 to dimerize and bind PtdSer on apoptotic cells, in a Ca 2+ -dependent reaction.GAS6/PROS1 initiate the phagocytic process by acting as bridging ligands between the apoptotic target, and the TAM receptors on the phagocyte.Ligand binding drives receptor dimerization, cross-phosphorylation of the tyrosine domains, and signal transduction via two distinct cascades leading to phagocytosis and anti-inflammatory signaling.Focal adhesion kinase (FAK), or Phospholipase Cγ (PLCγ) through phosphokinase C (PKC), drive CRKII/ELMO/ DOCK180-dependent activation of RAC1, in turn mediating actin cytoskeleton rearrangement necessary for phagocytic cup formation and engulfment of the target particle.Simultaneously, through the activation of the JAK/STAT kinases that block TLR, and the inhibition of NF-kB, the TAMs signal for the release of anti-inflammatory and pro-resolving cytokines.TAM-mediated anti-inflammatory signaling diverges from that of phagocytosis and involves nuclear events.Created with BioRe nder.com.inflammation, and on a daily basis for organs with high cell turnover such as the testes and retina.The testis and retina are tissues with elevated rates of cell turnover at steady state and cannot afford to mount an inflammatory response on a regular basis.
The term "phagocytic synapse" was coined by Goodridge et al.
to describe the point of attachment between the phagocyte and an AC to be engulfed, by analogy to the "immunological synapse" describing the specific requirements of contact between an antigen presenting cell (APC) and a T cell. 49Yet, two key differences exist between the immunological and phagocytic synapses: First, the immunological synapse is necessary to locally concentrate the soluble cytokines needed for effective signaling, whereas the phagocytic synapse is mainly generated by membrane-bound molecules, and it is still unclear whether a physical enclosure is necessary for successful phagocytosis.Second, the immunological synapse is necessary to cage the secreted cytokines and assists in increasing their local concentration to overcome a threshold necessary for a fruitful engagement.By contrast, phagocytosis is mediated by membranebound molecules, and therefore, there is no need to concentrate the effector molecules.Finally, the immunological synapse does not usually culminate in the uptake of another cell moiety.Nevertheless, the semblance of the immunological and phagocytic synapses in facilitating both signaling and cell-cell communication is significant.
As phagocytic receptors, the TAMs are part of the "phagocytic synapse," which is composed of (1) externalized phosphatidylserine (PtdSer), exposed by the moiety to be engulfed (ACs, myelin, synaptic boutons, photoreceptor outer segments); (2) the professional or non-professional phagocyte, expressing one or more TAM receptors on its outer membrane; (3) the TAM ligands GAS6 and/or PROS1, which act as bridging molecules indirectly linking the phagocyte and target moiety, and promote phagocytosis by stimulating TAMdependent intracellular downstream signaling (Figure 1). 1,9,265][56][57] As mentioned above, the gamma-carboxylation of the Gla residues occurs posttranslationally, and is necessary for Ca 2+ docking and the conformational change to catalyze PtdSer binding, as well as for TAM receptor activation.Ligands with non-carboxylated Gla domains, or Gla-less ligands where the Gla residues were altogether truncated, still allow for ligand binding, but not for activation of TAM receptors. 15,16On the carboxy-terminal, the SHBG domains of the TAM ligands bind to the Ig domains of their receptors, inducing their dimerization, and cross-phosphorylation of the intracellular tyrosine residues (Figure 1).TAM receptor activation triggers intracellular signaling cascades leading to actin cytoskeletal rearrangements necessary for formation of the phagocytic cup, as well as for the final internalization of the target particle. 1,26Phagocytosis is, like many other biological systems, a multi-step process that constitutes an important regulatory mechanism to avoid off-target phagocytosis, ensure that only particles exposing "eat-me" signals take part in the phagocytic synapse, and ultimately that the TAMs are fully activated to efficiently clear and degrade whatever must be engulfed.Interestingly, a rigorous density of PtdSer must be externalized on the particle membrane to fully activate the TAM system. 58Once TAMs initiate phagocytosis, a simultaneous series of anti-inflammatory signaling cascades are initiated in the phagocytes.Such TAM-dependent anti-inflammatory pathways were shown in dendritic cells (DCs) and macrophages, shifting their pro-inflammatory profile to an overall anti-inflammatory one.[61] It is noteworthy to mention that while PtdSer is required for recognition of the apoptotic particles, its exposure alone is not sufficient to promote engulfment by macrophages.Segawa et al. generated viable cells which overexpress and present PtdSer at comparable levels to those of ACs.They showed that these viable cells-exposing PtdSer to the same level of ACs-are not recognized by macrophages and not internalized until apoptosis was induced in these cells (Figure 2). 62ese results point to additional factors other than PtdSer, which allow macrophages to identify and recognize ACs, avoiding massive off-target phagocytosis of viable cells.On the contrary, even exposure of PtdSer in the presence of TAMs and their ligands would not guarantee engulfment.For example, bacteria and fungi present PtdSer, which was shown to promote their virulence, 63 but TAM-expressing immune cells encountering such pathogens do not exploit TAMs for bacterial or fungal uptake and elimination (Figure 2). 64,65Ligand availability is not assumed to be a limiting factor, as TAM ligands effectively function to activate TAM receptors in tissues and in vitro in the presence of other PtdSer-presenting particles.Moreover, TAM receptors expressed on the surfaces of macrophages, lymphocytes, endothelial cells, microglia, and many other cell types which are continuously exposed to TAM agonists are not constitutively activated, indicating this pathway is tightly regulated.Part of this regulation occurs at the ligand level by means of biochemical modifications, such as the as the vitamin K dependent γ-carboxylation of Gla domains.
Another biochemical modification that is thought to lend specificity in the context of ACs is ligand oxidation and oligomerization.Uehara and Shacter showed that upon binding to PtdSer, PROS1 undergoes oxidation of cysteine residues, and this reaction promotes formation of intermolecular disulphide bonds and oligomerization of PROS1.This sequence of events leads to more effective MERTK activation on macrophages. 66Together, the requirement for a series of complex and localized post-translational modifications contributes to the regulation and fine tuning of TAM signaling.It may be reasonable to speculate that additional unknown factors guide the decision of whether or not to activate TAM signaling (Figure 2).

| TAMS COUPLE PHAG O C Y TOS IS WITH ANTI -INFL AMMATORY S IG NALING
Among the multitude of cellular functions they supervise, the TAMs are also infallible participants of immune homeostasis maintenance and restoration.The TAM system lies in between the innate and adaptive immunity, suppressing the build-up of immune responses by releasing tissue repair mediators. 60One of the anti-inflammatory activities of the TAMs is clearance of ACs, 64,[67][68][69] which would induce inflammation if the cell corpses remain in the tissue.Evolution has provided TAM signaling with the ability to simultaneously promote efferocytosis by signaling cytoskeletal rearrangements to form the phagocytic cup as well as to curb the inflammatory response through the release of the anti-inflammatory and resolving cytokines IL-4, IL-10, IL-13, and TGFβ. 3,59,70This way, the same receptor mediates two seemingly independent cellular functions, which are in fact coordinated to maintain immune homeostasis and tissue health.At the molecular level, Tibrewal et al. found that for MERTK, the phosphorylation of tyrosine residue 867 (MERTK Tyr867 ) controls cytoskeletal rearrangements that are required for phagocytosis.Mutating tyrosine 867 abrogated engulfment, but did not affect MERTK-dependent anti-inflammatory function following LPS stimulation, thereby providing a molecular basis for dissociating engulfment from antiinflammatory functions.This study further demonstrated that the MERTK-dependent anti-inflammatory pathway is a post-nuclear event, involving transcriptional suppression of NFκB-dependent responses. 71Thus, the active inhibition of inflammatory signaling, as well as efferocytic signaling by TAMs are distinct both functionally and at the molecular level.Engulfment-associated cytoskeletal rearrangement also depends on MERTK phosphorylating and activating focal adhesion kinase at Tyr861 (FAK Tyr861 ), driving its binding to the intracellular domain of the β5 subunit of integrin αvβ5.Like MERTK, integrin αvβ5 functions as a transmembrane engulfment receptor, activated by MFGE8 (Milk Fat Globule and EGF Factor 8) which, like GAS6 and PROS1 binds PtdSer on ACs, essentially functioning as a bridging ligand. 72MERTK also induces p130 CAS phosphorylation in a αvβ5-dependent manner, to further activate the downstream F I G U R E 2 Biochemical regulation and specificity of TAM-mediated phagocytosis.Engulfment of different PtdSer-expressing particles employs distinct signaling molecules which have opposite effects, and are tailored to their specific biological contexts.The "eat-me" signal PtdSer functions in a versatile manner and is able to trigger different biological responses in phagocytes, depending on the type of target that exposes PtdSer, and the desired physiological outcome.Under healthy conditions (left), TAMs mediate engulfment of apoptotic cells (ACs) by phagocytes in a mechanism that is biochemically tightly regulated.The post-translational γ-carboxylation on the Gla domain of GAS6 and PROS1, and the presence of calcium ions lead to PtdSer binding and ligand dimerization, and is also necessary for TAM receptor activation.Further, it was shown that PtdSer induces oxidation of PROS1 cysteine residues, leading to oligomerization through disulphide bond formation.These biochemical (and possibly additional unidentified) modifications provide local regulation and allow for TAM ligands to bridge and bind the apoptotic moiety and the phagocyte, while activating TAM signaling at the same time.TAM activation in phagocytes not only leads to clearing ACs, but also activates anti-inflammatory signals, which help to maintain homeostasis, suppressing elevated immune responses.However, when the PtdSer-expressing molecules are expressed by infectious agents (right) such as bacteria or fungi, TAMs are not engaged.Instead, the toll-like receptors (TLR) and other pro-inflammatory responses are recruited, activating the immune system, even in the presence of TAM receptors and ligands.Created with BioRe nder.com.
While the anti-inflammatory function can be triggered by TAMs without prior particle internalization, 24,74 it is not clear whether AC uptake in vivo would necessarily be coupled to and stimulate anti-inflammatory signaling.Furthermore, whether phagocytosis of different moieties such as ACs, myelin, the outer segments of photoreceptors, or apoptotic bodies would result in similar antiinflammatory engagement remains unknown.Rothlin et al. showed that in DCs, the anti-inflammatory function of AXL is dependent on the type I interferon receptor and STAT1 (IFNAR/STAT1) signaling, highlighting that TAM heterodimerization with non-TAM receptors can influence TAM function. 24Future research may reveal that heterodimerization with other receptors, or interactions with other modifying proteins present in their vicinity in lipid rafts may guide in steering downstream TAM pathways.In this respect, AXL also signals as a heterodimer with epidermal growth factor receptor (EGFR), promoting drug resistance and survival in head and neck cancer. 75en triple KO mice lacking all TAM receptors were first reported in 2001, these mice exhibited severe autoimmunity and hyper-inflammation, even in the absence of experimental inflammatory stimulus. 28However, more recent studies have found that some of these spontaneous inflammatory phenotypes, such as arthritis, are not as prominent, 76 possibly due to improved cleanliness of modern institutional vivariums.This suggests that TAM receptors act in response to specific inflammatory stimuli rather than constantly regulating inflammation.In agreement, most in vitro studies have investigated the TAM anti-inflammatory function using prestimulated cells.For instance, CpG, poly(I:C), or LPS are commonly used in vitro to prime DCs and macrophages, to demonstrate TAM activation dampens inflammation.This enforced the concept that TAMs come into action mediating their anti-inflammatory function following a stimulus that has breached homeostasis.However, the notion that TAMs may continuously function "backstage" to regulate inflammation even at steady state and without any prior stimulation is supported by two recent independent studies.Maimon et al. showed that ablating PROS1 from macrophages was enough to abstain MERTK activation in naïve macrophages in vitro, rendering BMDMs hyper-inflamed at the cell level.This seems to be the case also in vivo, where ablation of PROS1 from myeloid cells led to inflamed lungs in otherwise healthy, non-challenged mice. 74A more recent report by Mercau et al. (further discussed below) revealed that MERTK inactivation leads to retinal inflammation even before the onset of high retinal turnover, indicating that in the absence of prior challenge, continuous action of TAMs is needed in order to maintain homeostasis.Moreover, because the high retinal turnover commences only later in life, this early time window allowed to differentiate between the anti-inflammatory and phagocytic functions of TAMs in vivo. 77

| TAM S I G NALING IN HOMEOS TATIC PHAGOC Y TOSIS
In a human body, several hundred billion cells undergo programmed cell death, or apoptosis, on a daily basis, in the effort of getting rid of old, poorly functional, or simply superfluous cells. 72This type of cellular death is preponderant in the homeostatic turnover of tissues, and beneficial for making room for younger and healthier cells. 52,72Life-long, high cell turnover routine processes include spermatogenesis, adult neurogenesis, and hematopoiesis, when millions of cells become apoptotic before completing their differentiation into mature sperm cells, neurons, and immune cells, respectively.In order to prevent disease, ACs must be approached and engulfed by tissue-resident phagocytes, which are also in charge of suppressing the inflammatory response at the same time. 78The importance of TAM-mediated phagocytosis to the healthy maintenance in these tissues is discussed in the next sections and summarized in Figure 3.

| TAM -MED IATED PHAG O C Y TOS IS BY S ERTOLI CELL S
An impressive apoptotic rate is observed in the epithelium of the seminiferous tubules of the testes, where spermatogenesis yields hundreds of millions of immature germ cells, of which less than onethird complete maturation and become functional spermatozoa. 79e entire spermatogenesis process is sustained by the multifunctional Sertoli cells, which "nurse" the spermatogenic cells throughout their development by maintaining hormonal and ion balance, providing nutrition and metabolism regulation. 80Importantly, the Sertoli cells are also the phagocytes specific to the testes.They engulf those germ cells that present abnormalities and failed to differentiate, as well as foreign antigens. 81,82One Sertoli cell is often in contact with 30-40 germ cells at different developmental stages.[84] Unsurprisingly, efficient phagocytosis by Sertoli cells is fundamental for healthy spermatogenesis and male fertility, 79,81,82 and this mechanism is driven by the TAM receptors in a PtdSer-dependent way. 27,81,85In the testis, Sertoli cells express all three TAM receptors while another cell type in the testes, the Leydig cells, are the source of the ligands GAS6 and PROS1. 27,86This creates a network of cell interactions that includes Sertoli, Leydig, and germ cells, and maintains the correct functionality of the male reproductive system (Figure 3). 87ong the first observed phenotypes of TAM-dependent faulty phagocytosis was male infertility. 1,27 Approximately 10 7 sperm cells per gram of testicular tissue are produced daily, of which around 75% die before reaching maturity.While necessary to maintain a balanced sperm Sertoli cell ratio, 79,83 the products of such massive apoptosis must be rapidly cleared to avoid release of toxins and reactive oxygen species which would affect healthy spermatogenesis. 81,88The simultaneous deletion of all three TAM receptors causes infertility due to their inactivation in Sertoli cells.In mice lacking functional TAM signaling, seminiferous tubule cell architecture is damaged, sperm and spermatogenesis are absent, testes are shrunk and present heightened inflammation characterized by increased infiltration of T-cell lymphocytes and inflammatory macrophages (Figure 4). 88,89As uncleared apoptotic cells accumulate in the testes, certain phenotypes progressively worsen over time.These include inflammation, the presence of autoantibodies against germ cell antigens, and the breach of the blood-testicular barrier.Zhang et al. characterized the time course of the inflammatory phenotype within the testis of TAM-deficient mice. 88Notably, these inflammatory and autoimmune features were absent prior to apoptotic spermatid accumulation, suggesting that inflammation is a secondary reaction to the failed clearance of apoptotic germ cells. 88Interestingly, single or double ablation of any combination of TAMs is not enough to cause sterility, indicating that the three receptors are either functionally redundant, or must work together to maintain spermatogenesis. 27,89However, F I G U R E 3 TAM-mediated phagocytosis in homeostasis.A schematic summary of unceasing phagocytic events undertaken by TAM signaling to maintain steady state.Depicted are the variety of phosphatidylserine (PtdSer)-presenting phagocytic targets in different tissues and their corresponding TAM-expressing phagocytes in the context of their ultimate biological function, along with the reporting studies.Synaptic pruning by microglia and astrocytes, as well as POS trimming differ, as they present engulfment of portions of live cells, whereas the other events involve the uptake of bona fide apoptotic elements.RPE, retinal pigment epithelium, POS, photoreceptor outer segments.Created with BioRe nder.com.
ablating Mertk together with either Axl or Tyro3 (Tyro3 −/− Mertk −/− or Axl −/− Mertk −/− ) is enough to cause a major reduction in testis weight and sperm counts.This suggests that MERTK may be the dominant TAM member mediating Sertoli cell phagocytosis, though its ablation alone is not sufficient by itself to cause infertility. 27,89A gene expression array performed in murine Sertoli cells deficient for all TAMs revealed that compared to TAM-expressing controls, Cd36 was among the significantly downregulated genes, and found to be implicated in phagocytosis by Sertoli, 90 as well as other cell types. 91,92Together with N-cadherin, an important mediator of Sertoli cell-spermatid adhesion, 93 these genes were identified as the main candidates to contribute to infertility. 89The discovery of the critical role fulfilled by TAMs and their ligands in the testes paved the road for further studies on their mediation of phagocytosis in other organs.

| TAM -MED IATED PHAG O C Y TOS IS IN HEMATOP OIE TIC ORG ANS
The thymus and bone marrow are another relevant example of organs with elevated cell turnover.Massive apoptotic cell death occurs in the developing thymus, in a process known as negative selection of the T cells. 94,957][98] Though to a lesser extent, the negative selection in the thymus continues for a small window of time postnatally, as the organ reduces its size and function.0][101] The TAM system, although prevalently regulating adult homeostatic mechanisms, 1 was recently found to be critical for the clearance of the negatively selected thymocytes during murine postnatal development (Figure 3).Jimenez-García and colleagues demonstrated that loss of MERTK and AXL results in decreased phagocytic activity of macrophages, resulting in dramatic accumulation of dead T cells.Furthermore, Axl/Mertk double mutant mice display a 50% reduction in F4/80 + CD11b lo macrophages, which normally express MERTK and are highly efferocytic in the bone marrow and thymus, impairing apoptotic thymocyte clearance. 61These macrophages are also responsible for continuously phagocytosing erythrocytes and their heme-iron components.In the absence of AXL and MERTK, the remaining macrophage subpopulations downregulate engulfment molecules such as CD163, further aggravating their phagocytic inefficiency. 61These data highlight the previously unrecognized role of TAM signaling in maintaining the health and function of hematopoietic organs.

| TAM -MED IATED PHAG O C Y TOS IS BY RE TINAL PI G MENT EPITHELIAL CELL S IN THE E YE
In the retina, phagocytosis is performed by the retinal pigment epithelial (RPE) cells.In addition to their phagocytic properties, for which RPE are considered professional epithelial phagocytes, they undertake numerous functions necessary for retinal viability. 102wever, instead of engulfing ACs, RPE cells ingest and metabolize the outer segments of viable photoreceptors (Figure 3).The photoreceptor outer segments (POS) constitute the outermost layer of the retina, responsible for light absorption. 103This phagocytosis is programmed to occur daily with light onset on a circadian basis, throughout life.POS are subjected to high intensity light during the day, inducing photo-deterioration of proteins and lipids and generating toxic oxidative products of phototransduction.3][104] Lu et al. were the first to report that TAM triple mutant mice were blind due to massive photoreceptor degeneration (Figure 4). 27A couple of years later, Mertk was appointed as the sole accountable gene causing visual loss in the Royal College of Surgeons (RCS) rat model of retinitis pigmentosa, 105 as well as in mice, 30,105 and humans. 106,107MERTK was found to be expressed on the apical microvilli of RPE cells, where it functions as an essential regulator of POS phagocytosis.TYRO3 acts similarly; however, its deletion alone is not enough to cause blindness, and its complete expression depends on MERTK levels. 108With respect to the ligands, the observation that only the removal of both GAS6 and PROS1 recapitulates Mertk −/− phenotypes was a major breakthrough in the TAM field for two main reasons: First, it provided the very first in vivo evidence of PROS1 being a functional ligand of TAMs, ending a hot debate as to the relevance of PROS1 as a TAM agonist. 14,35,36Second, GAS6 and PROS1 seem to act interchangeably, but are absolutely required for POS engulfment by RPE cells, and for maintaining retinal F I G U R E 4 Ramifications of TAM-mediated phagocytosis in disease.(A) In neurodegenerative disorders, phagocytosis through the TAMs has different implications.In Alzheimer's disease (AD), microglial engulfment of Aβ plaques results in the formation of dense-core plaques in the phagocytes, which are either unable to process them, or die and release the load.On the contrary, in Parkinson's disease (PD), phagocytosis promotes the clearance of α-synuclein protein aggregates, and absence of TAM signaling speeds up Lewy bodies formation in the substantia nigra.In multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), defective myelin uptake exacerbates autoimmunity and inflammation, which inhibits remyelination and aggravates disease progression.(B) In the retina, retinal pigment epithelium (RPE) cells trim the outer segments of rods and cones through TAMs.Ablating TAM receptors results in early elevated RPE inflammation and degeneration of photoreceptors, leading to vision loss in mice and retinitis pigmentosa in humans.(C) In the tumor microenvironment, elevated cell turnover attracts TAM-mediated phagocytosis by macrophages, but also activates anti-inflammatory agents, thereby creating conditions favorable for tumor growth and progression.(D) In hypercholesterolemic (HCL) conditions, TAMmediated LDL uptake leads to lipid accumulation in macrophages that turn into foam cells and drive the formation of atherosclerotic plaques in blood vessels.This mechanism is further aggravated by immune cell activation, infiltration, and accumulation, leading to atherosclerosis, and in severe cases to myocardial infarction.(E) Systemically, altered phagocytosis due to TAM loss of function leads to accumulation of cellular debris, persistent inflammation, and autoimmune disorders, as in systemic lupus erythematosus (SLE).Absence of TAM signaling was found to develop into Lupus-like clinical manifestation in mice.(F) In a mouse model of peripheral nerve injury, Schwann cells perform autophagy by using the TAMs to uptake degraded myelin.(G) Sertoli cells of the testes utilize the TAMs to phagocytose unviable sperm cells during spermatogenesis.Disruption of such signaling leads to apoptotic sperm cells accumulation, reduced testes size, and infertility.Created with BioRe nder.com.homeostasis. 31The involvement of TAMs in RPE-mediated uptake of ROS diverts from the mainstream theme where TAMs uptake ACs which expose PtdSer.However, the molecular basis remains, with the outer segments of photoreceptors locally externalizing PtdSer in a circadian rhythm, thereby providing PtdSer availability for PROS1 and GAS6 to bridge between the POS presenting PtdSer and the TAM-expressing RPE in a viable cell. 109Hence, TAM function in the retina diverges from their traditional role in efferocytosis (Figure 3).
Retinitis pigmentosa is a group of genetic disorders leading to the degeneration of rod and cone photoreceptors.As a result, light cannot be detected and transformed into visual information. 110is leads to peripheral blindness, which progressively advances to tunnel vision and central vision loss. 111,112Mertk is highly expressed by RPE cells, 108 and is a major regulator of POS phagocytosis. 105,107,108,113The retinal degeneration associated with MERTK mutants observed in rat, mice, and humans shaped the concept that failed phagocytosis of POS leads to retinal degeneration.For this reason, retinitis pigmentosa in MERTK-deficient patients has been largely attributed to the impaired phagocytosis by RPE.However, not all rodents and humans carrying mutations in the Mertk gene showed similar blindness severity, and in some cases showed no phenotype at all, hinting at the possibility that genetic variants of Mertk, or potentially of Tyro3 which is also expressed by RPE may modify the disease course. 114,1155][116] Two new Mertk mutants that were generated (Mertk −/−V2 and Mertk −/−V3 ) did not exhibit retinitis pigmentosa, unless crossed to Tyro3 mutant mice (Mertk −/−V2 Tyro3 −/−V2 mice), identifying TYRO3 as a disease modifier affecting early-onset photoreceptor degeneration. 114,115major breakthrough in understanding the role of Mertk in the pathophysiology of retinal degeneration was recently made by Mercau et al., after observing elevated inflammation in the RPE of Mertk −/−V2 mice as early as postnatal Day 10 (P10), a time point at which there are no signs of retinal degeneration, and, most importantly, phagocytosis of POS is minimal.In their work analyzing P10 mice, Mercau et al. show that MERTK deficiency causes early and unresolved inflammation manifested by monocytes infiltration and pronounced microglial activation. 116Moreover, when RPE phagocytosis is severely inhibited in integrin β5 null mice, no gross retinal degeneration was observed, 117 suggesting that lack of phagocytosis alone is not sufficient to drive retinal degeneration, and pointing to the observed RPE inflammation as a potential cause for retinal degeneration (Figure 4).The authors hypothesized that if inflammation would underlie retinal degeneration in Mertk −/−V1 mice, then pharmacological treatment curbing this inflammation is expected to ameliorate retinal degeneration.To test their speculation, Mercau

| TAM -MED IATED PHAG O C Y TOS IS IN THE B R AIN
Although accounting for only 10%-15% of total brain cells, microglia are the first-line guardians of the central nervous system (CNS). 118[122] Microglia function as sentinel cells and continuously scan the brain parenchyma both in homeostasis and disease. 118,123In homeostatic conditions, microglia are busy phagocytosing newborn neurons that do not complete their maturation, and are therefore excluded from developing functional synapses.Indeed, among all the neurons that are generated during neurodevelopment and adult neurogenesis, only a fraction is incorporated within the neural networks. 124The remaining undergo apoptosis and are rapidly cleared by microglia. 119,124,125A similar mechanism is observed in the adult brain, when neurogenesis decreases significantly, but is retained and confined in two neurogenic niches, the subgranular zone of the hippocampus, and the subventricular zone, lateral to the ventricles. 126,127This clearance of apoptotic newborn neurons by microglia is mediated by AXL and MERTK in both adult neurogenic niches (Figure 3). 128Moreover, mature synapses are continuously remodeled by experience and learning, sometimes generating connections that are no longer used. 129,130Supernumerary synapses are therefore eliminated as synaptic bodies that are engulfed by microglia in order to maintain proper signal transduction, and strengthen new or existing neural circuits. 131,132Microglia are commonly defined as the macrophages specific to the CNS, and although during acute inflammation peripheral monocytes infiltrate the brain and become morphologically indistinguishable from the local resident microglia, these two populations still retain genetic and molecular differences. 133In the attempt to identify such differences, Butovsky et al. performed gene expression analysis and reported that the TAM signaling genes Gas6, Pros1, and Mertk were significantly enriched in murine microglia compared to peripheral monocytes.Similarly, these three genes were exclusively expressed by human adult, and, though to a lower extent, fetal microglia, and absent in other immune cells, 134 suggesting the dominance of TAM signaling in regulating different aspects of microglial function.Microglia use MERTK to ingest synapses, in a mechanism that is not only confined to the mere digestion of the synapse, but in turn negatively regulates neurogenesis through their phagocytic secretome, creating a feedback loop that ultimately regulates adult hippocampal plasticity and maintains brain homeostasis (Figure 3). 135croglia are the professional phagocytes for the CNS, 118 but are not the only cell type performing phagocytosis in the brain.
Interestingly, astrocytes have been reported to engulf synapses too. 1369][140] Synapse elimination by astrocytes occurs during development and in adulthood, and is driven by the PROS1/ MERTK pathway, in parallel to the MEGF10 pathway, also initiated by PtdSer.
Inhibition of both pathways had a stronger effect on astrocyte engulfment, indicating these two PtdSer pathways are distinct and function in parallel, allowing for developmental and adult synaptic pruning and neural network refinements 136 (Figure 3).
Another important source of PtdSer in the brain is myelin, a lipidrich membrane ensheathing neuronal axons, both protecting axonal tracts and allowing for rapid movement (saltatory conduction) of the neural impulse. 141Free myelin results from disrupted synapses, dead neurons, and the degeneration of the myelin-producing oligodendrocytes.The clearance of such debris is crucial to promote homeostatic synapse refinement and synaptogenesis.The presence of PtdSer in myelin licenses myelin as membrane targets for TAMmediated phagocytosis in the CNS. 142,143Similarly, the Schwann cells and oligodendrocytes of the peripheral nervous system (PNS) use the TAM pathways to phagocytose myelin, though more in an injury context rather than during homeostasis (Figure 4). 144seases in the nervous system generate degraded myelin, as in the case of the autoimmune disease multiple sclerosis (MS) (Figure 4).MS patients produce autoantibodies against their own myelin sheaths, 145 progressing with infiltration of T cells, activation of microglia and astrocytes, and further entry of monocytes. 146mune responses lead to inflammation and demyelination, loss of oligodendrocytes, and degeneration of axonal and motor neurons, [146][147][148] also culminating in accumulation of myelin debris.0][151] The professional phagocytes in the inflamed brain comprise of the local proliferating and activated microglia, as well as monocyte-derived macrophages (MDMs) that infiltrate the CNS from the vasculature. 118,123,152,153rtk expression was altered in MS patients' myeloid cells, resulting in defective myelin phagocytosis by MDMs and a pro-inflammatory environment in human brains. 143,154Consistent with decreased MERTK activation, lower levels of PROS1 were found in the plasma of MS patients. 155MERTK-mediated engulfment of myelin by human microglia was maximal following their exposure to TGFβ, also accompanied by a significant upregulation of MERTK, PROS1, and GAS6.Myelin uptake also led to reduced inflammation through the release of IL-10, 143 pointing to mechanistic similarities to the uptake of ACs. 1568][159] In line with these findings, administration of GAS6 enhanced clearance of myelin debris and improved remyelination. 158These findings are of high relevance for the amelioration of MS symptoms: The pharmacological manipulation of MERTK to exploit its anti-inflammatory activity through phagocytosis may be employed to boost remyelination in MS patients.The challenge will be to find an effective treatment to promote debris clearance while maintaining the right balance between immune stimulation and suppression.
A common pathological hallmark of neurodegenerative disorders is the aggregation of insoluble and indigestible oligomers and larger protein aggregates, which, if left uncleared, contribute to the development of chronic inflammation and oxidative stress, driving mechanisms of neurodegeneration. 160,161Alzheimer's disease (AD) (Figure 4) is a neurodegenerative disorder characterized by the progressive accumulation of such insoluble protein oligomers and aggregates known as Amyloid beta (Aβ) plaques.Patients show dementia, memory loss, and inability to communicate and perform daily tasks. 161Although the contribution of Aβ plaques to AD is still under debate, 162 their association with disease focuses effort on understanding their role in AD pathogenesis.Aβ plaques are enriched in exposed PtdSer, 163 anticipating a role for the TAM ligands and receptors.AXL has been associated with AD pathogenesis: RNA-sequencing (RNA-seq), and single-cell RNA-seq (scRNA-seq) revealed upregulated Axl expression in microglia associated with human Aβ plaques and in the microglia of the 5xFAD murine AD model, respectively. 164,165AXL's soluble ectodomain (sAXL) bound to GAS6 in the cerebrospinal fluid (CSF) is a biomarker and prognostic factor in AD patients. 166Moreover, using the APP/PS1 mouse model of AD, Huang and colleagues demonstrated that microglia employ the TAM system to detect and phagocytose Aβ plaques, mainly through MERTK and AXL, with GAS6 as the putative bridging ligand.Axl −/− Mertk −/− microglia are unresponsive to Aβ fibrils, lacking recognition, proliferation and motility orientation, and ultimately also lack phagocytic clearance. 163Contrary to expectations, the authors observed that faulty TAM-mediated microglial phagocytosis of Aβ correlated with the deposition of fewer dense-core plaques. 163is unexpected observation may be explained by three independent studies which show that functional microglia contribute to Aβ plaque compaction: The first reports that Aβ engulfed by microglia are released as packed clusters to the extracellular space due to toxicity and death of the microglia, and this deposition contributes to Aβ plaque growth. 167Two additional studies find that microglia depletion reduces accumulation of Aβ plaques, 168,169 thus supporting an active role for TAM-mediated microglial phagocytosis of Aβ in AD pathology.PROS1 was recently identified as a novel microgliaderived biomarker in the hippocampi and serum of AD mice, also found at elevated levels in the sera of AD patients. 170It is not clear whether PROS1 is involved in AD pathology or is upregulated as part of an intrinsic anti-inflammatory and repair mechanism to restore homeostasis.More research is needed to clarify how the involvement of TAMs and their ligands in amyloid plaque engulfment and in regulating inflammation is linked to AD.
Parkinson's disease (PD) (Figure 4) is another neurodegenerative disease where alpha-synuclein (α-SYN) forms small soluble oligomers and larger aggregates within subcellular inclusions called Lewy bodies. 171Inflammation and gliosis also come into play in PD, often with deleterious consequences, 172 raising the question of impaired TAM function.In a mouse model of PD, induced by the introduction of human α-SYN in Nrf2 −/− mouse brains, disease progressed as α-SYN aggregates accumulated.This was accompanied by microglial inflammation and attenuated expression of Mertk and Axl. 172other study in mice which utilized a late-onset hereditary form of PD, driven by a point mutation of the human α-SYN gene (SNCA A53T ) reports the upregulation of both AXL and its soluble form (sAXL), documented to be markers of inflammation. 128Interestingly, such upregulation was exclusively coupled with an increase in expression of the microglial activation marker IBA1, especially in the spinal cord, but also in the brain.Survival of PD mice carrying the SNCA A53T mutation was slightly extended following Axl and Mertk ablation.This was explained by the possible TAM-dependent phagoptotic engulfment, worsening the disease. 128

| THE TAM RECEP TOR S PERFORM PHAGOP TOS IS
In the previous paragraphs, the importance of TAM-dependent phagocytosis in preventing secondary necrosis, elevated inflammation, and the development of serious disorders was discussed.
Nonetheless, like many biological mechanisms, phagocytosis must be regulated to avoid uncontrolled cell clearance, including that of viable cells.As a general rule, phagocytosis is secondary to initiation of cell death, as externalization of the classic eat-me signal PtdSer is induced by apoptotic pathways. 51However, possibly due to a subtoxic insult, live cells may express "eat me" signals, or lose the expression of "don't eat me" signals, promoting their engulfment.This type of primary phagocytosis was characterized for the first time by Brown et al., and named phagoptosis.Phagoptosis may lead to the clearance of stressed, but otherwise viable cells, and further exacerbate disease development. 173For example, after brain ischemia, the affected regions suffer from low-oxygen levels and excessive glutamate release, which cause extensive neuronal death. 174In such a scenario, uptake of those dead neurons within the area of ischemia is crucial, and beneficial for tissue repair.In the neighboring areas, however, neurons may be stressed, yet functional.In such subtoxic conditions, cells can transiently expose PtdSer, 175 and be engulfed alive, causing severe neuronal loss. 174Surprisingly, sparing these stressed, but viable neurons from clearance has proved to be beneficial, with rats showing reduced motor deficits and overall improved outcome in a brain ischemia/stroke model. 176The transient upregulation of Mertk in microglia and macrophages at the same time phagoptosis occurred, suggested MERTK may be involved in this process.Consistently, the uptake of stressed neurons was reduced by 60% in Mertk mutants, which showed reduced brain atrophy and improved motor functions.In parallel, in vitro studies demonstrated that Mertk mutant microglia engulfed a smaller number of glutamate-stressed neurons.Taken together, these data suggest that MERTK-mediated phagoptotic cell clearance may constitute a new driver of brain pathology. 176Therefore, inhibiting MERTK to prevent the engulfment of stressed, yet viable neurons may help the recovery after mild ischemia.Similarly, Fourgeaud et al., showed that ACs clearance through the TAMs not always prevents disease development and point to phagoptotic engulfment of stressed, but live motor neurons by AXL and MERTK as a possible mechanism worsening PD symptoms, and speeding animal death. 128In conclusion, the TAM receptors are components of the phagocytic mis-targeting that characterizes phagoptosis.Therefore, exploiting the TAM system at specific physiological contexts is an interesting therapeutic approach to improve symptoms and prevent neurodegeneration.

| C ARDIOVA SCUL AR DIS E A S E S
Atherosclerosis is the most frequent type of cardiovascular diseases (CVD) and leading cause of death worldwide. 177It is caused by the accumulation of cholesterol in the arteries, creating masses of inflamed lipid material that builds up atherosclerotic lesions.With time, these become big enough to obstruct the blood flow leading to myocardial infarction (MI) and stroke. 177,178Both tissue-resident and monocytederived macrophages play an important role in the development, or recession, of atherosclerotic plaques. 177When low-density lipoprotein (LDL) levels are high (hypercholesterolemia, HCL), tissue-resident macrophages are unable to handle the heavy load of lipids through canonical pathways (autophagy or storage), leading to lipid aggregation and atherosclerosis initiation.The overloaded tissue-resident macrophages try unsuccessfully to discharge the lipid overload, and become so called "foam cells", 179 which subsequently undergo apoptosis and necroptosis. 177,180In the early stages of this process, efferocytosis by other macrophages prevents plaque growth.However, in later stages, foam cells downregulate Mertk, rendering efferocytosis inefficient (Figure 4). 181As a consequence, MDMs are recruited by pro-inflammatory stimuli, and atherosclerotic plaques grow. 177,181afting Mertk +/+ or Mertk −/− BM into atherosclerotic mice showed that macrophages derived from Mertk −/− BM were unable to efficiently clean up ACs, increasing necrosis and speeding up lesion development. 182,183Similarly, Knock-down (KD) of Mertk in a model of advanced atherosclerosis showed increased formation of necrotic plaques due to defective MERTK-dependent efferocytosis of ACs. 184chanistic studies suggested that tissue-resident macrophages may be unable to perform efferocytosis in advanced stages of lesion formation due to their increased expression of Ca 2+ /calmodulin-dependent protein kinase γ (CaMKIIγ), which suppresses ATF6, a transcription factor regulating liver X receptorα (LXRα), which is a Mertk-inducing transcription factor.This study reveals a macrophage CaMKIIγ/ATF6/ LXRα/MERTK pathway as key in development of cardiovascular disease, and highlights that MERTK inactivity not only leads to ACs accumulation, but also prevents the phagocytosis-dependent release of resolving cytokines. 185This pathway has been recently targeted in a pre-clinical model study by Tao et al.: Introducing small interfering RNA (siRNA) nanoparticles targeting murine CaMKIIγ, Mertk expression in macrophages increased.This resulted in efficient ACs phagocytosis and reduced necrotic plaques, constituting a promising targetable pathway. 186In human patients, the titer of the soluble form of MERTK (sMERTK) may serve as a marker for cardiovascular disease, as it was found to positively correlate with necrosis of plaques and symptoms typical of ischemia and stroke. 183Taken together, these studies highlight the importance of MERTK-mediated efferocytosis within atherosclerotic plaques, and identify novel therapeutic targets to prevent atherosclerosis progression.

| SYS TEM -WIDE IMPLI C ATI ON S OF DEFEC TIVE TAM RECEP TOR-MED IATED PHAGOC Y TOSIS
Given that the TAM receptors are implicated both in clearing phagocytic cells in numerous organs, and in engulfing different moieties, some implications of TAM-driven phagocytosis are system-wide.This is the case for autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren syndrome (SS).SLE is characterized by chronic inflammation triggered by defective clearance of ACs in the lymph nodes germinal center, and the production of autoantibodies against nuclear antigens and double-stranded DNA.It affects several different organs including skin, joints, brain, blood vessels, lungs, and kidneys 187 (Figure 4).Predictably, TAM receptor KO mice develop lupus-like clinical manifestations, including systemic autoimmunity and enlarged lymph nodes. 28,67Plasma levels of secreted forms of AXL and MERTK (sAXL and sMERTK, respectively), as well as GAS6, are reported to be increased in adult onset SLE patients, and to correlate with renal symptoms severity, specifically glomerulonephritis. 188,189Similarly, sMERTK levels, although not sAXL, sTYRO3 and GAS6, were found to correspond to active disease in Juvenile SLE (JSLE) patients. 190Elevated levels of the cleaved forms of TAM receptors imply they may bind their ligands and function as competitors with cell-bound TAMs, inhibiting phagocytosis. 1913][194] Taken together, these data on human patients show that sTAM levels in serum may serve not only as new biomarkers for SLE activity and prognosis, but also represent great therapeutic targets. 191RA is the most common autoimmune disorder, manifesting itself as chronic synovial fluid inflammation, and resulting in cartilage and bone damage. 195TAM receptors are expressed in several locations of the synovial tissue.Akin to SLE pathogenesis, recent findings similarly revealed that sTYRO3, sMERTK and sAXL levels are elevated in the synovial fluids of RA patients.However, no differences were found in GAS6 expression. 196,197Interestingly sTYRO3 levels positively correlated with increased secretion of proinflammatory cytokines in the joints, identifying TYRO3, among the other TAM components, as an important potential candidate promoting RA pathogenesis and severity. 196In SS, autoimmunity affects salivary and lacrimal glands, where elevated apoptosis and defective efferocytosis are observed.Monocytes isolated from SS model mice exhibit decreased MERTK signaling and defective efferocytosis, with elevated levels of sMERTK in plasma, and similar inflammatory profiles to Mertk −/− mice. 198Despite the lack of evidence of TAMs involvement in SS, several studies reported low levels of TAM receptors mRNAs in mononuclear cells of SS patients' blood, together with high sMERTK and autoantibodies titter. 199Interestingly, monocytes isolated from SS patients were reported to perform inefficient efferocytosis compared to those from healthy controls, hinting at their involvement in SS through defective cell clearance and reduced inhibition of IFN signaling. 200Such conditions contribute to defective efferocytosis and a pro-inflammatory environment that ultimately exacerbates autoimmunity.
If the TAM anti-inflammatory ability is favorable in the context of most pathologies discussed so far, it is not always beneficial in cancer.The tumor microenvironment (TME) is characterized by high cell turnover and is therefore abundant with PtdSer-expressing cells: intra-tumor ACs, but also viable PtdSer-expressing tumor cells and tumor-associated endothelial cells. 57,201PROS1 and GAS6 are attracted to the high presence of PtdSer, bridging between PtdSer-expressing cells and TAM-expressing phagocytes, promoting phagocytosis and anti-inflammatory signaling, altogether favoring tumor growth (Figure 4).Phagocytosis through the TAMs shifts the pro-inflammatory status of macrophages secreting TNFα, IL-6, IL-1β, and nitric oxide (NO), which are strong activators of the immune response, to anti-inflammatory, known to secrete resolving cytokines (IL-4, IL-10, IL-13, TGFβ).These events provide for a tumorsupportive environment and feed its growth. 57,74,202,2035][206][207][208] It is likely that there is a positive selection for PtdSer-expressing cells in the TME, allowing these cells to survive and contribute to tumor survival and promote epithelial to mesenchymal transition (EMT), as demonstrated in mouse models for breast, 209 brain, 210 and lung cancers. 74Given that efferocytosis is anti-inflammatory, 48,211 TAM-driven clearance of ACs within tumors may contribute to an immune-suppressed and cancer-promoting environment.Thus, pharmacological treatments tackling cancer should therefore aim at inhibiting TAM-mediated phagocytosis in the TME. 57

| CON CLUDING REMARK S
TAM receptors and their ligands are often functionally redundant, as mentioned above.Yet, this pathway accurately initiates a multitude of intracellular signaling cascades, each within its specific physiological context.As briefly mentioned, TAMs also initiate additional non-phagocytic cellular functions such as proliferation, migration, and cell fate decisions with implications ranging from stem cell biology to cancer signaling. 2,57,206In this respect, how TAMs acquire their functional specificity in a variety of biological contexts is still not fully understood.Now, we know that ACs are not the only targets recognized and engulfed by TAMs, as PtdSer-exposing live cells, or portions thereof (synapses, POS), as well as myelin, are also engulfed by phagocytes through TAMs.This way, the same system is employed to clear up different particles that may affect tissue health, and simultaneously dampen the inflammatory response.Disruption of TAM signaling causes particle accumulation and heightened inflammation, resulting in tissue degeneration, as shown in many organs including the brain, retina, testes, joints, and blood vessels.On the contrary, TAMphagocytosis is not necessarily beneficial in the tumor and stroke micro-environments.However, TAMs are not universal executers of phagocytosis, as they are not activated by bacteria or fungi, even if all components seem to be in place (Figure 2).How then is this specificity regulated?Additional open questions are whether TAMs similarly recognize the different PtdSer-exposing moieties, regardless of their size and content?Engulfment alone introduces quite a few challenges to the phagocyte.Following recognition of the target, uptake requires cytoskeletal changes to generate the phagocytic cup leading to internalization.Once internalized, digesting a bulk of biomass is probably challenging, especially since ACs carry toxic waste products.In this respect, Anwar et al. showed that MERTK also promotes survival of macrophages under oxidative stress conditions, mimicking AC uptake. 212Despite many similarities to phagocytosis of pathogeninfected ACs, efferocytosis is anti-inflammatory.Remarkably, TAMs converge cytoskeletal rearrangement, anti-inflammatory signaling, and survival pathways.Linking survival and anti-inflammatory signaling to phagocytosis in general, and to efferocytosis in particular, is evolutionarily beneficial, as it allows optimization of cell resources and shortens the response time.Moreover, activation of protective pathways safeguards from the potentially detrimental consequences of engulfing the toxic cargo, enabling successful accomplishment of efferocytosis.This is particularly important for postmitotic phagocytes such as RPE and Sertoli cells.
Understanding the specificity of TAM function is only beginning to be revealed.While MERTK mainly functions at steady state, AXL is recruited following stress induction, 213 but how this is regulated is still unknown.Initial reports point to functional variability among the ligands in regulation of adult neural stem cell proliferation, 2 and in POS phagocytosis in the retina. 214reover, in order to cope efficiently with the toxic products of cell death, evolution generated different families of receptors, other than the TAMs, to perform phagocytosis in a PtdSer-dependent way. 2157][218] Whether the many PtdSer receptors that have evolved are unique or functionally interchangeable was addressed by Penberthy et al., who asked whether BAI1 overexpression would compensate for MERTK loss of function in the testis and retina.Interestingly, although BAI1 overexpression was able to rescue ACs build-up in the testes, it was not sufficient to rescue photoreceptor degeneration in the retina. 215The authors hypothesized that this difference is due to the fact that the uptake of POS by RPE is not a traditional corpse clearing event, as is apoptotic spermatid clearance.However, a more recent research now revealed that retinal degeneration in Mertk −/− mice is due to early inflammation rather than lack of POS uptake. 116Nevertheless, given that both BAI1 and MERTK have anti-inflammatory functions when dealing with homeostatic clearance of ACs, 78,219 these pathways are clearly distinct and not completely redundant.
Similarly, clearing degraded myelin by the two PtdSer engulfment receptors MERTK and MEGF10 was not redundant. 136Such experiments reveal that despite significant molecular and mechanistic similarity, TAMs and other phagocytosis receptors are not simply redundant, or interchangeable, illuminating functional difference and hence the need for numerous PtdSer-recognizing phagocytosis receptors.
By virtue of the expression on virtually every cell and tissue, TAM receptors and their ligands are major regulators of adult homeostasis, though they also play important roles during development.Their quintessential homeostasis-related functions are efferocytosis, discovered in the nineties, and anti-inflammatory signaling.Clearly, the development of therapeutics targeting the TAMs is of high interest and has great potential to treat diseases involving system-wide, or organ-specific, implications.However, to best harness this system, different aspects of TAM-mediated phagocytosis still remain to be elucidated.
et al. characterized inflammation in the RPE of Mertk −/− Tyro3 −/− deficient mice, and found elevated pro-inflammatory intracellular pathways culminating with the release of interleukin-6 (IL-6), type I and II interferons (IFNs) and tumor necrosis factorα (TNFα), which are mediated by the Janus kinase 1/2 (JAK1/2).Indeed, treating these mice with the anti-inflammatory drug Ruxolitinib, a JAK1/2 inhibitor, partially protected the retina from degeneration, further validating inflammation as the driving cause of the retinitis pigmentosa-like retinal degeneration observed in Mertk −/− mice. 116Thus, rather than improving phagocytosis, targeting the mediators of inflammation which are activated in the absence of TAM signaling should be investigated as a better approach and important therapeutic line to help retinitis pigmentosa patients.Hence, by characterizing the different time points at which inflammation and phagocytosis occur, Mercau et al. show that the retina provides an in vivo model to segregate the anti-inflammatory function of MERTK from its phagocytic role, and provides new evidence for a net anti-inflammatory role for MERTK until POS engulfment becomes evident, at P10.