ID wrote the article and generated the figures. NB, JM, MV, and AR wrote the article and critically appraised the figures.

#### FUNDING

MV acknowledges funding from the Biotechnology and Biological Sciences Research Council (BB/M025160/1). AR is

#### REFERENCES

part funded by a UK Medical Research Council (MR/K013386/1). NB acknowledges funding from the EPSRC and MRC Centre for Doctoral Training in Optical Medical Imaging, OPTIMA (EP/ L016559/1).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Barth, Marwick, Vendrell, Rossi and Dransfield. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Chronicles of Cell Death Foretold: Specificities in the Mechanism of Disposal

*Lindsey D. Hughes1†, Lidia Bosurgi 2,3†, Sourav Ghosh4,5\* and Carla V. Rothlin1,4\**

*1Department of Immunobiology, School of Medicine, Yale University, New Haven, CT, United States, 2 I. Medizinische Klinik und Poliklinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany, 3Bernard-Nocht-Institut für Tropenmedizin, Hamburg, Germany, 4Department of Pharmacology, School of Medicine, Yale University, New Haven, CT, United States, 5Department of Neurology, School of Medicine, Yale University, New Haven, CT, United States*

Massive turnover of cells occurs through apoptosis during the constant remodeling of

#### *Edited by:*

*Amiram Ariel, University of Haifa, Israel*

*Reviewed by: Ariel Munitz, Tel Aviv University, Israel Yi Wu, Temple University, United States*

#### *\*Correspondence:*

*Sourav Ghosh<EMAIL_ADDRESS>Carla V. Rothlin<EMAIL_ADDRESS>
> *† Co-first authors.*

*The title is inspired by the Gabriel Garcia Marquez novel "Chronicle of a death foretold."*

#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 24 September 2017 Accepted: 23 November 2017 Published: 11 December 2017*

#### *Citation:*

*Hughes LD, Bosurgi L, Ghosh S and Rothlin CV (2017) Chronicles of Cell Death Foretold: Specificities in the Mechanism of Disposal. Front. Immunol. 8:1743. doi: 10.3389/fimmu.2017.01743*

our tissues at homeostasis, from the shedding of cells at exposed barrier surfaces to the elimination of autoreactive lymphocytes. However, a surge of apoptotic cells also accompanies tissue damage, infection, and inflammation. A salient feature of apoptosis in either scenario is the exposure of phosphatidylserine (PtdSer) on the outer leaflet of the plasma membrane. In response to this cue, a range of phagocytes are charged with the sizeable task of engulfing apoptotic bodies and disposing of the billions of cells that perish each day. The presence of apoptotic cells in the remarkably distinct immunological settings described above, therefore, raises the question of how phagocytes are able to coordinate appropriate responses to apoptotic cells—from their silent removal to the production of growth factors or tissue repair molecules—following such a ubiquitous signal as PtdSer exposure. Here, we consider several emergent properties of phagocytes and apoptotic cell clearance that may facilitate specification among this suite of potential responses.

Keywords: apoptosis, homeostasis, tissue repair, phagocytic receptors, signal integration

#### INTRODUCTION

"…but in this world nothing can be said to be certain, except death and taxes." (Benjamin Franklin)

Apoptotic cell death is a perennial component of tissues from embryonic development through to adulthood. Is death the great equalizer in which all apoptotic cells are handled identically by phagocytes? Or is every instance of apoptosis unique—the trigger and context of a particular cell death event granting specificity to the phagocyte response? During many instances of developmental apoptosis, such as digit formation or postnatal pruning of neuronal circuits, there is simply a need to cull. Conversely, in the case of apoptosis following tissue damage or injury, there is a fundamental need to replace the dying cells; disposal must be linked to regenerative signals. Viral infection of cells can also trigger death by apoptosis, such that the indiscriminate uptake of this cell could threaten the phagocyte with infection. Does knowledge of why cells are undergoing apoptosis enable us to foretell the nature of their removal? Are there distinct mechanisms of identification in effect to enable this functional diversity? In this review, we discuss the view that the array of phagocytes, their repertoire of receptors, the temporal expression pattern of these receptors, and coincidence detection mechanisms may confer specificity to the disposal of dead cells.

### PHAGOCYTE AND RECEPTOR DIVERSITY: REDUNDANT OR REQUISITE?

### Molecular Basis of Apoptotic Cell Recognition

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 damageassociated 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.

# Dynamics of Phagocytic Receptor Expression

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 thioglycollateinduced 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 αMβ2 expression by J774 murine macrophages (25). Instead, exposure to each of these inflammatory stimuli mediated activation of RAP1, a Ras-like GTPase that enhanced αMβ2 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).

#### Signaling an Appropriate Response

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 αvβ5 and MERTK, have also been shown to engage the ELMO-1/Dock-180 machinery (27, 28), with constitutively active MERTK only phosphorylating p130CAS, an adaptor protein in this pathway, if αvβ5 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.

#### Phagocyte Identity and Localization

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 steadystate 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.

## SPECIFICATION THROUGH INTEGRATION

Phagocytes rarely are exposed to a single stimulus at once nor, as considered, do they react equivalently to a given signal under all circumstances. The integration of multiple, contemporaneous signals therefore represents a general mechanism through which cells could generate distinct, tailored outputs in response to their microenvironment. This integration could occur at the molecular and/or cellular level, and then serve to further regulate the immune response, such as through amplification or inhibition of specific effectors.

One example of discrete signals combining to generate a specific output by phagocytic macrophages is the synergy between the sensing of apoptotic cells and of type 2 cytokines. Upon infection or injury, there is a significant increase in the magnitude of apoptotic cell death, with resident cells damaged by the insult dying off and infiltrating immune cells turning over. Multiple soluble ligands, like cytokines, are also produced at different phases of the inflammatory response, and phagocytes are also capable of sensing these factors. Work from our laboratory established that BMDMs, as well as macrophages in the lung, the intestine, the visceral adipose tissue, and the peritoneum, launch an efficient tissue repair response only when the sensing of IL-4/IL-13 occurs in the presence of apoptotic cells (**Figure 1**) (40). Importantly, this was not merely an additive effect of two parallel pathways—the presence of apoptotic cells alone did not induce the expression of some of the remodeling-associated genes analyzed. Integration

of these two signals was shown to be dependent on the detectors AXL and MERTK*,* two phagocytic receptors expressed at both steady state and/or upon damage by resident and monocytederived macrophages. Genetic ablation of *Axl* and *Mertk*, or the administration of Annexin-V, which coats and obscures exposed PtdSer on apoptotic cells, were each sufficient to curtail this specialized tissue repair genetic program in macrophages in response to IL-4/IL-13. These findings demonstrate that apoptotic cell sensing can influence phagocyte activity in ways beyond its described immunosuppressive effect.

Interestingly, while the maximal induction of remodelingassociated genes like *Arg1*, *Retnla*, and *Chil3* were reliant on macrophages receiving these two signals, other IL-4-mediated responses were not. Gene set enrichment analysis of BMDMs treated with IL-4 alone identified categories such as pattern recognition signaling pathways, regulation of cytokine production, chemotaxis, and the defense response, all of which were unaffected by an additional treatment of the BMDMs with exogenous apoptotic cells. Like other cytokines, IL-4 and IL-13 are produced at multiple phases of the immune response and have pleiotropic effects, including driving cell proliferation, antigen presentation, antimicrobial activity, and this induction of tissue remodeling (41–43). Collectively, these results shed light on a role for apoptotic cells in specifying the action of IL-4 on one of its target cell populations. In this way, the requirement of both signals functions as a checkpoint, restricts the expression of repair factors to both an appropriate time and location, and helps to avoid the potentially detrimental effects of aberrant remodeling. While the effects of this signal integration have been characterized, further investigation is needed to determine the precise molecular players that interact downstream in the IL-4/ IL-13 and TAM signaling pathways.

It is important to note that additional tissue-specific factors have also been described to promote the resolution program in phagocytes in response to IL-4. Minutti *et al*. recently identified surfactant protein-A (SP-A) and complement component C1q as secondary stimuli that enhance the proliferative and tissue-remodeling capacity of IL-4-activated macrophages from the lung and the peritoneal cavity/liver, respectively (**Figure 1**) (44). In support of this, SP-A or C1q were each necessary for extensive repair responses in settings of acute and chronic damage, like with *Listeria monocytogenes* infection or in the Dineal PD-4 model of peritoneal fibrosis. *Sp-a*−/− mice, for example, exhibited severe immunopathology in the lung and increased worm burden in the intestine following infection with the helminth *Nippostrongylus brasiliensis* in comparison to their WT counterparts. Integration of these pathways is at least partially mediated through myosin 18 A (Myo18A); *in vivo* delivery of IL-4 was shown to increase the expression of this shared receptor for SP-A and C1q in macrophages isolated from the lung, liver, and peritoneal cavity (44).

As referenced before, uptake of apoptotic cells by phagocytes has been shown to suppress the response to inflammatory stimuli, like TLR ligands (45–51). Engulfment of apoptotic cells

tailored effector functions. Consistent with this proposition, in vertebrates, not only are the number of phagocytic receptors and ligands expanded but also their engagement is integrated with specific signals from the microenvironment.

by immature murine bone marrow-derived DCs was shown to dampen the response to LPS, with specific reduction of IL-12 secretion and CD86 expression, but not of other proinflammatory cytokines or costimulatory molecules, like TNFα and CD40 (50). In a similar manner, ingestion of apoptotic corpses by LPS-stimulated human or murine macrophages diminished secretion of proinflammatory molecules TNFα and IL-1β, but also enhanced release of anti-inflammatory signals, such as TGF-β1 and PGE-2 (45, 48, 49, 51). Thus, beyond suppressing proinflammatory events, integration of TLR signaling and apoptotic cell sensing actively promotes the generation of an anti-inflammatory environment.

The precise mechanism of integration of TLR signaling and uptake of apoptotic cells still needs to be fully elucidated. Intracellular lipid sensors have been described to contribute to this immunosuppressive effect of apoptotic cells (52, 53). Additionally, integration of phagocytic receptor and TLR signaling with cytokine signaling has been characterized as another strategy for dampening the inflammatory response by phagocytes. The receptor tyrosine kinase AXL, which is highly expressed by DCs (20, 30), was found to physically associate with the R1 chain of the type I IFN receptor (IFNAR1) upon administration of its ligand Gas6 (30). Engagement of and interaction between these two receptors led to the activation of STAT1, which in turn triggered SOCS1 and SOCS3 to limit both cytokine and TLR signaling (30). This cooperation highlights how the pairing of apoptotic cell sensors with other detectors not only can promote a specialized transcriptional program, but also can negatively regulate phagocyte activation in a specific manner. The extent to which phagocytic receptor signaling integrates with other cytokine pathways, and whether these are broader features of all phagocytic receptors, remain to be fully explored.

### CONCLUDING THOUGHTS

The functional diversification of apoptotic cell removal in mammals is consistent with the evolutionary expansion of PtdSer receptors. While efferocytosis itself is conserved from *C. elegans* to mammals, at least some of the mammalian receptors such as BAI-1, TIM4, and the TAMs do not have orthologs in

#### REFERENCES

*C. elegans.* The argument that increased redundancy of these receptors simply ensures efficient removal of dead cells, as the risk for autoimmunity increases with evolution, cannot be formally ruled out. Intriguingly, TAM, integrin, and MEGF10 mediated efferocytosis require "opsonization" of the apoptotic cargo with their respective ligands, including PROS1/GAS6, MFGE8, or C1q (54) (**Figure 2**). This system seems analogous to that seen in *Drosophila* hemocytes, in which secreted Mcr, Tep II, or Tep III opsonize *Candida albicans*, *Escherichia coli*, or *Staphylococcus aureus*, respectively, during phagocytosis (55). The selective advantage of such a system is difficult to understand simply on the basis of redundancy; rather, it favors a paradigm wherein layers of specificities can be built in. Overall, evolutionary expansion of phagocytic receptors may have enabled cargo-selective disposal and the resulting specification in phagocyte response. Further inquiry of this topic may permit the establishment of a "code" (56), through which knowledge of the various input signals from the dying cell and the surrounding microenvironment can predict the functional output of the phagocyte.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

### ACKNOWLEDGMENTS

The authors thank Macy Akalu and Silvina del Carmen for their helpful discussion and reading of this manuscript.

### FUNDING

This work was supported by grants from the National Institutes of Health (NIH-NIAID R01 AI089824 to CR, NIH-NCI R01 CA212376 to CR and SG), the Deutsche Forschungsgemeinschaft (grant SFB841 to LB), Alliance for Lupus Research (grant 332789 to CR), the Yale Immunobiology Department (NIH-NIAID T32 AI007019 to LH), and the National Science Foundation (DGE-1122492 to LH). CR is a HHMI Faculty Scholar (grant 55108561).

phagocytosis by Lp-PLA2. *Cell Death Differ* (2014) 21(5):825–35. doi:10.1038/ cdd.2014.1

by DNGR-1, a receptor for dead cells. *Immunity* (2012) 36(4):635–45. doi:10.1016/j.immuni.2012.03.008

infectious disease, and cancer. *Cell Death Differ* (2016) 23(6):962–78. doi:10.1038/cdd.2016.11

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Hughes, Bosurgi, Ghosh and Rothlin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Macrophage Clearance of Apoptotic Cells: A Critical Assessment

*Siamon Gordon1,2\* and Annette Plüddemann3*

*1College of Medicine, Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan City, Taiwan, 2Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom, 3Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, United Kingdom*

As the body continues to grow and age, it becomes essential to maintain a balance between living and dying cells. Macrophages and dendritic cells play a central role in discriminating among viable, apoptotic, and necrotic cells, as selective and efficient phagocytes, without inducing inappropriate inflammation or immune responses. A great deal has been learnt concerning clearance receptors for modified and non-selfligands on potential targets, mediating their eventual uptake, disposal, and replacement. In this essay, we assess current understanding of the phagocytic recognition of apoptotic cells within their tissue environment; we conclude that efferocytosis constitutes a more complex process than simply removal of corpses, with regulatory interactions between the target and effector cells, which determine the outcome of this homeostatic process.

#### *Edited by:*

*Amiram Ariel, University of Haifa, Israel*

#### *Reviewed by:*

*Dror Mevorach, Hadassah Medical Center, Israel Sylvain Perruche, Institut National de la Santé et de la Recherche Médicale, France*

*\*Correspondence:*

*Siamon Gordon<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 29 September 2017 Accepted: 16 January 2018 Published: 30 January 2018*

#### *Citation:*

*Gordon S and Plüddemann A (2018) Macrophage Clearance of Apoptotic Cells: A Critical Assessment. Front. Immunol. 9:127. doi: 10.3389/fimmu.2018.00127*

Keywords: apoptosis, apoptotic cells, clearance of apoptotic cells, phagocytosis, macrophages

### INTRODUCTION

Programmed cell death, apoptosis, is closely linked to recognition and clearance by phagocytosis, resulting in anti-inflammatory and compensatory growth responses during fetal and adult life. Prolonged or incomplete containment and destruction during the apoptotic process can merge into secondary necrosis to bring about pro-inflammatory and pathological responses by phagocytes that are still poorly understood. Many aspects of this process are covered in excellent reviews by leading investigators, including Henson (1), Arandjelovic and Ravichandran (2), Nagata and Tanaka (3), Lim et al. (4), and their colleagues, which should be consulted for detailed exposition. We have previously reviewed the process of phagocytosis in relation to infection and microbial recognition (5), emphasizing its cell biology in model systems. We focus here on clearance of apoptotic cells *in situ* by heterogeneous tissue mononuclear phagocytes, in health and disease.

# A BRIEF HISTORY

Although current nomenclature settled around the early reports by Wyllie et al. (6), the observations of cell death in its many manifestations date back to the nineteenth century, from descriptions

**Abbreviations:** AIP, apoptosis-induced proliferation; DC, dendritic cell; Gas 6; growth arrest specific protein 6; GPCR, G protein-coupled receptor; IECs, intraepithelial cells; IGF-1, insulin-like growth factor-1; KLF, Kruppel-like factor; LPS, lipopolysaccharide; LXR, liver X receptor transcription factor; MPS, mononuclear phagocyte system; MerTK, proto-oncogene c-mer tyrosine kinase; Megf8, milk fat globule—EGF factor 8 (lactadherin); MNGC, multinucleated giant cell; NET, neutrophil extracellular trap; PGE2, prostaglandin E2; ProS1, gene for protein S(eattle); PRRs, pattern-recognition receptors; PS, phosphatidyl serine; PPAR, peroxisome proliferator activator receptor transcription factor; ROS, reactive oxygen species; SLE, systemic lupus erythematosus; TAM, tyrosine receptor kinases Tyro 3, Axl and MerTK; TLR, toll-like receptor; TNF, tumour necrosis factor; tgf beta, transforming growth factor beta.

by Virchow, Metchnikoff, and many pathologists. Wallach et al. have provided an instructive time line of concepts of tissue injury and cell death in inflammation (7). An historic perspective of macrophages, phagocytic mechanisms, and lysosomal digestion is provided elsewhere (5, 8). The pre-eminent role of tissue macrophages in clearance was emphasized in the twentieth century, as a primary function of the reticuloendothelial system, subsequently renamed the mononuclear phagocyte system (MPS) (9). Studies in *Caenorhabditis elegans* (10) stimulated genetic dissection of apoptosis and clearance by epithelial cells in organisms that lack "professional" phagocytes; important discoveries of macrophage clearance followed in *Drosophila* and other model organisms, such as zebra fish and mice. Uptake of dead cells by non-professional phagocytes in vertebrates became overshadowed by emphasis on macrophages and related dendritic cells (DCs), although recent studies (2) have to some extent redressed the balance; turnover of photoreceptors by retinal pigment epithelia and of aberrant sperm in the testis by Sertoli cells are highly active functions of non-hematopoietic phagocytic cells, and uptake of cell corpses has also been demonstrated in epithelia, fibroblasts, astrocytes, and cancer cells, the so-called non-professional phagocytes (11). Different terms have emerged for a range of distinct though related processes, in addition to efferocytosis (12); these include necroptosis (13), pyroptosis (14), phagoptosis (15), ferroptosis (16), trogocytosis (17), and entosis (18), depending on one or other characteristic feature. Mevorach and colleagues have introduced clarity into the terminology of this expanding topic, which will be defined as relevant, below (19). Henson and Bratton (20) provided early evidence that clearance of programmed apoptotic cell death by macrophages gave rise to anti-inflammatory effects, unlike the pro-inflammatory consequences of the uptake of necrotic cells, which could follow at a further stage of programmed cell death, during infection or as a result of accidental injury. Another time line of particular interest in this area is given by Nagata and Tanaka, who pioneered the role of phosphatidyl serine (PS) and membrane lipid reorganization in the recognition of apoptotic cells (3).

The physiological role of apoptotic cell clearance by macrophages has been documented in organ development, tissue remodeling, e.g., in the uterus and mammary gland, repair and potential cell replacement following injury and, in a few species, regeneration of complex organs. In pathology, monocytes, macrophages, and DCs are important contributors to inflammation and its resolution, following disposal of necrotic corpses and subcellular constituents, e.g., during infection, innate and autoimmunity, atherogenesis, and malignancy. Many authors have considered cell death, its recognition, disposal, and regulation as central homeostatic functions of the MPS; aspects of this topic are reviewed by the various contributors to this Frontiers of Immunology collection, cited as available at the time of writing (21, 22). We shall not deal in this review with the mechanisms of cell death itself.

#### MONONUCLEAR PHAGOCYTES ARE HIGHLY HETEROGENEOUS

The cells of the mammalian MPS constitute a widely dispersed population derived from common hematopoietic progenitors, which are distinct in the embryo and adult (23). Tissue-resident macrophage populations in the fetus are distributed from yolk sac and fetal liver precursors from mid gestation, and turn over locally to a variable extent throughout adult life (24). From birth, bone marrow-derived monocytes are recruited to replenish and supplement tissue macrophage populations in the steady state, and in response to inflammatory, metabolic, infectious, and malignant disease processes, as required. Circulating mononuclear cells contain precursors of macrophages, DCs, and osteoclasts, and subpopulations of monocytes that are characterized by distinct marker antigens and receptors (25). Recent single cell RNA analysis has revealed additional mononuclear cell subpopulations in blood without functional characterization (26). Trahtemberg and colleagues identified two human blood monocyte-derived DC subpopulations which differ in their expression of surface markers, phagocytic clearance, and responses to the uptake of apoptotic cells (27). The markedly heterogeneous mononuclear phagocytes in tissues display the ability to phagocytose particulates to a variable extent, depending on their differentiation, recruitment, and activation by tissue-specific and microbial stimuli. The ability of neutrophils which are highly phagocytic for opsonized microbes, and of eosinophils, basophils, and mast cells, to phagocytose apoptotic targets has been less studied. In addition, MPS cells variably display fluid phase, macropinocytic, and receptor-mediated endocytosis, which contribute to their clearance of solutes and smaller particulates. In response to local need, resident macrophages can migrate from adjacent reservoirs, as elegantly demonstrated through *in vivo* experiments by the groups of Wang and Kubes and Robbins, respectively, in liver (28) and heart (29). The prototypic and organ-specific phagocytic activity of the mixed populations of macrophages in different organs differ considerably, as shown by elegant clearance experiments *in vivo*, described in detail below (30, 31). Fusion of monocytes and macrophages gives rise to the formation of osteoclasts in bone and of multinucleated giant cells (MNGCs) in granulomatous diseases (32). We have demonstrated a striking difference in the phagocytosis of a range of particulates, including latex and complement-, but not antibody-opsonized sheep erythrocytes by IL4/13 cytokine-induced MNGC (33). Finally, activation of macrophages by innate and Th1- and Th2-dependent adaptive immune stimuli induces a spectrum of changes in cellular phenotype, including cytocidal and phagocytic capacity (34), hitherto mainly studied *in vitro*.

### MYELOID CELL TURNOVER

Myeloid leukocytes themselves undergo apoptosis and necrosis, as well as inducing these processes in a range of target cells. We therefore briefly consider aspects of their own turnover, clearance, and mechanisms of death that may be relevant to subsequent consequences. Recent studies by Yona and colleagues have established the circulation time and precursor–product relationships of different human monocyte subsets (35). Macrophages can become long-lived, terminally differentiated tissue-resident cells which remain biosynthetically active while ceasing to proliferate, but recently recruited monocytes and activated macrophages and DC themselves can undergo apoptosis within days of responding to inflammatory and infectious stimuli. By comparison, neutrophils are short lived and readily proceed from apoptosis to secondary necrosis when activated during acute inflammation; eosinophils may survive longer in tissues than usually appreciated. The microbicidal and cytocidal activities of activated neutrophils and monocyte/macrophages depend on generation of reactive radicals derived from oxygen through a respiratory burst; in addition, degranulation by neutrophils and eosinophils releases potent neutral proteinases such as elastase, as well as myeloperoxidase, to initiate cytotoxicity, whereas interferon gamma activated macrophages contribute to target cell killing by release of nitrogen metabolites generated by inducible nitric oxide synthase. Other mechanisms of myeloid cell cytotoxicity include chromatin extracellular trap (neutrophil extracellular trap) production by neutrophils and probably activated macrophages and release of tumour necrosis factor (TNF)alpha. Inflammasome activation and IL-1 release depend on selected proteolysis by cytosolic caspases. Depletion of essential amino acids such as tryptophan and arginine in their local environment contribute to cell death.

### INTERACTIONS BETWEEN MACROPHAGES AND APOPTOTIC CELLS

We have outlined the heterogeneity of the macrophage populations involved in clearance, and the complexity of their potential apoptotic/necrotic cargo. It has become customary to break the overall process down into distinct stages: "find me" (36, 37), by chemokine-receptor attractants and plexin–semaphorin guidance/repulsion (38, 39); "eat me" (40) and "don't eat" me (41) signals between apoptotic cells and phagocytes, involving diffusible products, plasma components such as thrombospondin, and intercellular interactions. We focus here on recognition (42) by macrophages of well-defined apoptotic cells. Apart from the welldocumented chromatin and cytoplasmic changes, we consider PS (43) and other potential ligands expressed on the surface of senescent and apoptotic cells and the wide range of direct and indirect opsonic receptors expressed on the macrophage surface (**Figure 1**). This information is based mainly on *in vitro* studies, with some support obtained from mouse and other genetic models *in vivo*. Phagoptosis, the uptake of viable cells (15), resulting in their destruction, depends on exposure of "eat me" and/or loss of "don't eat me" signals; it mediates turnover of erythrocytes, neutrophils, and other cells, and also contributes to defense against infection.

Several aspects of our present understanding deserve further comment: there is a bewildering range of possible molecular interactions reported in this literature, depending to a large extent on the interests of the researcher rather than proven relative importance under different conditions *in vivo*. It is perhaps not surprising that so critical a housekeeping function should involve considerable redundancy; it is well known that inhibitors or ablation of favorite individual receptor–ligand pairs are at best only partial in their effect *in vitro*, and often undetectable *in vivo*. For example, this was found in our own studies of possible SR-A involvement in the thymus, a site of avid apoptotic thymocyte clearance by macrophages (44). Up to a point, one could argue that some of these candidates could act in a common pathway or be redundant. A more likely interpretation is that there is organ-specific involvement of particular receptor– ligand pairs, as indicated by new evidence, discussed in more detail below. Furthermore, most previous studies have failed to appreciate the modulation of phagocytic activity by physiological and pathological regulation of particular pathways in different tissue environments. We should expect wide variation from the experimental use of species as divergent as *Drosophila*, zebra fish, mouse, and man, with clearance mechanisms arising by convergent as well as divergent evolution. Finally, since different cell types can perform the same function, constitutively or after induction, it is essential to identify the particular cells involved by cell-specific deletion in any experimental model; for example, this was demonstrated by Ravichandran and colleagues in a receptor transgenic overexpression model which showed enhanced clearance was attributable to epithelial cells and not macrophages, as might have been assumed (45). The macrophage ablation studies during development, e.g., in PU-1 null mice (46) show remarkably little impact except for minor deficiencies in interdigital web regression; presumably, alternative mesenchymal cells can compensate for developmental clearance of apoptotic cells. Failure to clear erythrocyte nuclei, however, does create hemodynamic problems after birth and early death postnatally can also be ascribed to infection.

### MECHANISMS OF CLEARANCE OF APOPTOTIC CELLS BY MACROPHAGES

We have previously outlined some of the broad features of opsonic, antibody, and complement-mediated phagocytosis (5), the phagocytic synapse model based on Dectin-1 mediated uptake of yeast particles (47), and a kinetic exclusion model of other plasma membrane molecules during uptake (4); its relevance to the mechanism of apoptotic cell uptake has not been determined. Intravital microscopy has revealed striking recruitment of different mononuclear cell populations during development (48) and following sterile and infectious injury and repair (28). Phosphatidylinositol kinases play an important role in signaling and ingestion of apoptotic cells (49). Various phosphoinositide mediators derive from preformed membrane phospholipids and reorganization of lipids within the plasma membrane bilayer with resultant PS exposure on the outer surface, as elucidated by the groups of Nagata and Tanaka (3) and Kay and Grinstein (50). BAI-1, an adhesion G protein-coupled receptor (51, 52), mediates uptake of apoptotic cells *via* PS recognition, followed by Dock and Elmo signaling. Effects of apoptotic cell uptake on membrane traffic, vesicular fusion and fission, the role of small GTPases, phagosome formation and function (53), as well as possible interactions with autophagy require further investigation. Similarly, there is need for more studies on the effects of apoptotic cell uptake on gene and protein expression, for example, by *in situ* hybridization and multiplex histochemistry, as well as of posttranslational and epigenetic modifications, in heterogeneous cell populations and individual macrophages, *in vivo* and *in vitro*.

### CLEARANCE OF APOPTOTIC CELLS BY RESIDENT TISSUE MACROPHAGES *IN VIVO*

It has been known for some time that resident macrophage populations in different organs such as liver, gut, lung, bone marrow, spleen, and brain are heterogeneous in their morphology, turnover, and antigen expression profile. Gene expression studies by Lavin and colleagues have documented organ-specific gene and enhancer patterns, as well as canonical signatures for a range of tissue macrophage populations (54). Apoptotic cell clearance is mediated by distinct pathways in different organs and helps to shape the local phenotype, as shown by recent studies by the groups of Hidalgo (31) and Magarian Blander (30). A-Gonzalez and colleagues used a parabiotic model to study homeostatic leukocyte clearance *in vivo*. Distinct receptors, opsonins, and transcription factors were demonstrated in uptake by macrophages in the bone marrow, spleen, intestine, liver, and lung interstitium; alveolar macrophages in the lung were not accessed by blood delivery. Mutant mice deficient in milk fat globule—EGF factor 8 (lactadherin), liver X receptor transcription factor alpha/beta, peroxisome proliferator activator receptor transcription factor gamma, and proto-oncogene c-mer tyrosine kinase (MerTK) differed from wild-type controls and varied in their contribution to uptake in bone marrow, spleen, and liver. Annexin A1 and Timd4 were also utilized to determine tissue-specific dynamics and expression of different phagocytic mediators. Gene expression analysis demonstrated preservation of distinct core signatures, but phagocytosis imprinted a distinct anti-inflammatory profile, upregulation of CD163 and of CD206, the macrophage mannose receptor, and decrease of the pro-inflammatory expression of IL-1 beta. Expression of CD206 made it possible to identify phagocytic macrophages in the steady state in the absence of experimental manipulation. Clearance activity confirmed the diurnal rhythm of neutrophil turnover. Several interesting aspects of this study require further study: the role of variation in apoptotic rate in neutrophils, the impact of non-circulating apoptotic cells, e.g., generated in the thymus by dexamethasone treatment, and the absence of detectable annexin binding in some experiments, consistent with uptake and subsequent killing of viable cells by phagoptosis. Furthermore, the effects of concurrent inflammation and infection on the clearance of apoptotic as well as necrotic cells, add an additional layer of complexity.

The study by Cummings et al. used a different genetic strategy to study the effects of apoptosis in a single organ, the small intestine, on gene expression by macrophages and DC, after uptake of dying epithelial cells (30). Diphtheria receptor expression in mice was targeted to the intestine by a villin construct with an eGFP reporter; low dose diphtheria toxin induced synchronous apoptosis selectively to ileum epithelium without affecting barrier function. Five subpopulations of APC were isolated before and after apoptosis by FACS, and gene expression was analyzed. Two CD64+ macrophages and one CD24+ DC population were shown to display distinctive signatures after phagocytosis of apoptotic cells; the macrophage signatures included lipid metabolism and aromatic amino acid catabolism, whereas the DC subset program was consistent with activation of regulatory CD4+ lymphocytes. A common signature of suppression of inflammation consisted of distinct genes in macrophages and DCs. Only the DCs were induced by apoptotic intraepithelial cell (IEC) uptake to migrate to draining lymph nodes and found able to induce immunological tolerance. Several of the differentially induced genes in phagocytes overlapped with susceptibility genes for inflammatory bowel disease. CD103+CD11b+ and distinct CD11b+ only macrophages, which sampled the majority of apoptotic IEC+, upregulated growth arrest specific protein 6, Cd 163, and MerTK genes, whereas CD103+ DC expressed the gene for CD105, a member of the mannose receptor family. Other differentially expressed genes included those implicated in apoptotic cell clearance, enhanced anti-inflammatory, and reduced pro-inflammatory pathways.

A recent study by Roberts et al. (55) demonstrated that selected tissue-resident macrophages in the peritoneal and pleural cavities, and lung alveolar space, are locally programmed for silent clearance of apoptotic cells. Specific populations of macrophages clear apoptotic cells in different tissues; they share features that limit recognition of nucleic acids in response to signals from their local tissue microenvironment. The transcription factor Krüppel-like factor-2 (KLF2), and to a lesser extent KLF4, control the expression of many genes within the macrophage clearance program. These characteristics include high expression of receptors for apoptotic cells such as Tim4, but low expression of toll-like receptor (TLR)9 and reduced TLR responsiveness to nucleic acids. The environmental signals in different tissues, which are unknown, can override inflammatory stimulation and are lost when cells are isolated, *in vitro*. Other tissue-resident macrophages vary in their inhibition of inflammatory responses after apoptotic cell uptake, and it is unclear how homeostasis is maintained in the face of infection, or restored during resolution of inflammation.

Similarly, Baratin et al. reported that a population of CX3CR1+CD64+MerTK+CD11c+ resident macrophages, but not CD8alpha DC, were the main local efferocytic cells in the T zone of lymph nodes; however, these scavenger macrophages did not prime CD4 T cells efficiently, unlike DC (56). Tingible body macrophages, prominent in germinal centers in lymph nodes, spleen, and other lymphoid structures, clear apoptotic B lymphocytes. Their possible role in B lymphocyte affinity maturation and antibody diversification and interactions with follicular DCs and other mesenchymal stromal cells require further investigation (57).

### ROLE OF PHAGOCYTIC CLEARANCE OF DYING CELLS IN INFLAMMATION AND ITS RESOLUTION, TISSUE GROWTH, AND IMMUNE REGULATION

The uptake of apoptotic cells by macrophages is not "silent," but anti-inflammatory. Secretory mediators include cytokines [transforming growth factor beta (tgf beta), IL-10] and arachidonate metabolites, e.g., prostaglandin E2, as well as a range of proresolving mediators. Addition of macrophages that have ingested early apoptotic cells, to lipopolysaccharide (LPS)-induced inflammatory cells *in vitro* or *in vivo*, reduced inflammatory pathways (20), whether through the above mediators or by undefined direct contact mechanisms. However, genome wide gene expression, proteomic and metabolomic studies may yield a more complex picture of pro- and anti-inflammatory signature markers which should be reinvestigated by newly developed population and single cell methods. *In vitro* studies need to take into account the tolerogenic and pro-inflammatory subtypes of macrophages and DC, the nature of the apoptotic cell targets, clearance receptor usage, and possible association with TLR. Previous exposure to priming agents, including apoptotic cells themselves, can convert the response to a secondary challenge (58). The *in vivo* studies described above have provided evidence for "imprinting," comparable to "trained immunity" (59), of an anti-inflammatory phenotype in several different tissues, but further studies are needed in the presence of infection or trauma, and associated cell death.

Studies in *Drosophila* (48) and a range of vertebrates have implicated macrophages in the replacement of epithelial and other cells during development and in tissue repair after wounding. Apoptosis and proliferation of microglia are coupled in the adult brain (60). Dying cells themselves are able to generate recruiting and mitogenic signals to attract and interact reciprocally with macrophages. These include a range of trophic factors such as insulin-like growth factor-1 (IGF-1) (45) and TNF-related cytokines (61), released after phagocytic clearance. Extracellular reactive oxygen species (ROS) produced by epithelial disk cells drive apoptosis-induced proliferation *via Drosophila* macrophages (61). The Ravichandran group has shown that IGF-1 released by macrophages in response to uptake of apoptotic cells, can inhibit apoptotic cell clearance by neighboring epithelial cells, while enhancing their uptake of smaller vesicles (45). Ueno et al. showed that CX3CR1+ microglia were required for survival of selected cortical neurons during development, ascribed to IGF-1 production after uptake of apoptotic neurons (62). Conversely, Brown and Neher have postulated that microglial phagoptosis mechanisms contribute to neuronal loss, synaptic sculpting, and loss of neuronal processes during development and neurodegeneration (63).

A striking example of the link between phagocytic clearance by macrophages and cell growth and differentiation was demonstrated by Kawane et al. (64). DNAse II in macrophages is responsible for destruction of nuclear DNA from erythroid precursors after enucleation, and essential for definitive erythropoiesis in mouse fetal liver. Recent studies by Fearnhead et al. have shown that Ferroptosis, a regulated physiological cell death process which requires free ferrous iron, occurs through Fe(II)-dependent lipid peroxidation when the reduction capacity of a cell is insufficient (16). Most studies have not distinguished between apoptotic, anti-inflammatory and immunomodulatory, versus pro-inflammatory, necrotic targets, or the source of contact-dependent, vesicular, or soluble products such as ROS and lipid metabolites. The nature of the apoptotic stimulus, for example, programmed versus X-ray-induced cell death, the heterogeneity of mononuclear phagocyte populations, and the local tissue environment can all contribute to the outcome. Most striking is the regenerative capacity of species such as axolotls (65) after limb amputation, for example, which depends on early recruitment and engulfment by phagocytic macrophages (66). A recent comprehensive analysis of gene expression during regeneration of the axolotl limb provides a resource to identify critical genes (67).

Dying cells release a complex mixture of lipids, oxidized phosphoryl choline derivatives, which can display opposing activities, interfering with TLR signaling to prevent inflammatory responses, while hyperactivating DCs and macrophages to promote inflammation by IL-1. CD14, the microbial LPS receptor, expressed by monocytes and macrophages, and downregulated by DC, is an early central regulator of both activities (68). Zanoni et al. demonstrated further that isolated lipid constituents of the mixture induce inflammasome-dependent macrophage hyperactivation *via* CD14, without compromising macrophage viability, thus promoting inflammation in sepsis, but not lethality.

IL-4 and other Th2 cytokines, possibly of innate sources, induce growth and an alternative activation pathway of macrophages, which has been implicated in wound repair (32). The macrophage mannose receptor (CD206), one of the characteristic signature genes induced by IL-4, is upregulated in mouse models of intestinal apoptosis, in which there is regeneration of epithelium, although it has not been determined whether it is required. A recent study has shown that apoptotic cell phagocytosis selectively amplifies the gene signature induced in macrophages by IL-4/13 (69). The tyrosine receptor kinases Tyro 3, Axl, and MerTK (TAM) receptors and their ligands play distinct immunoregulatory roles in DCs and macrophages (70) (**Figure 2**). Tyro3 and gene for protein S(eattle) mediate a negative feedback loop between DC and activated Th2 lymphocytes to inhibit type 2 immunity (71); by contrast, Mer-TK and Axl on macrophages require a local apoptotic ligand to promote a

full tissue repair program (69). If lysosomal peptides gain access to MHC or cytosol, cross-presentation can induce autoreactive CD8+ cytotoxic lymphocytes. Ly6+ monocytes are able to efferocytose apoptotic cells and cross-present cell-associated antigens to CD8+ T cells; this is enhanced by ligation of TLR7, but not TLR4 (72). Autoreactive Th17 lymphocytes can also be induced by apoptotic cell products in the context of microbial infection (73). Kleinclauss et al. reported that intravenous infusion of apoptotic spleen cells induced a tgf beta-dependent expansion of regulatory T lymphocytes, contributing to tolerance, induced by macrophages and not DCs (74). Saas and colleagues (21) have discussed the mechanism of early apoptotic cell infusion to prevent and limit ongoing autoimmune inflammation.

#### CONCLUDING REMARKS

Given the importance of the biological process of apoptotic cell clearance, it seems surprising that it might go wrong so infrequently; is there such redundancy and backup to keep it safe, or are we looking in the wrong place? Of course, we know of some autoimmune conditions, e.g., systemic lupus erythematosus (SLE), to which inefficient apoptotic cell clearance contributes; Munoz et al. (75) have discussed the role of nucleic acid and auto-antibody complexes in the pathogenesis of recurrent inflammation, resulting in production of type I interferon, a hallmark of SLE. A major effort is under way to use monoclonal antibodies directed against CD47, a "don't eat me" signal expressed by selected malignancies, to promote tumor cell ingestion by macrophages (40). A promising, safe protocol has been developed for

#### REFERENCES

adoptive transfer of mononuclear phagocytes that have started to engulf apoptotic cells, prophylactically, to facilitate allogeneic hematopoietic transplantation (76). It will be important to characterize the appropriate subpopulation of macrophages for such therapeutic applications. Conversely, we have good reason to believe that uptake of apoptotic HIV-infected lymphocytes by macrophages can provide a route to infection of healthy CD4+ T cells (77).

Although the research efforts in this area are mainly based on insights of several decades ago, it would be advisable to use the more advanced methods now available to revisit the fundamental aspects of the phagocytic process. It is exciting to see the recent adoption of intravital methods to study gene expression and growth responses *in vivo*. The holy grail might be to extend the limited regenerative capability of hematopoiesis, intestinal epithelium, and liver more widely to other tissues. In this regard, it would be wise to use a broad comparative approach to discover the factors that shut regeneration down during evolution. Or would that risk enhancing malignant diseases (78)?

#### AUTHOR CONTRIBUTIONS

SG conceived and wrote the manuscript. AP reviewed and edited the manuscript and designed the figures.

#### ACKNOWLEDGMENTS

We thank colleagues who have sent us preprints of their current work.

identification of limb regeneration factors. *Cell Rep* (2017) 18:762–76. doi:10.1016/j.celrep.2016.12.063

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Gordon and Plüddemann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Apoptotic Cell Clearance in *Drosophila melanogaster*

*Qian Zheng1 , AiYing Ma1,2, Lei Yuan1 , Ning Gao1 , Qi Feng1 , Nathalie C. Franc3 \*† and Hui Xiao1 \*†*

*1Key Laboratory of the Ministry of Education for Medicinal Plant Resources and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource Development of Endangered Crude Drugs in the Northwest of China, College of Life Sciences, Shaanxi Normal University Xi'an, Xi'an, China, 2College of Biological Sciences and Engineering, Beifang University of Nationalities, Yinchuan, NingXia, China, 3Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, United States*

The swift clearance of apoptotic cells (ACs) (efferocytosis) by phagocytes is a critical event during development of all multicellular organisms. It is achieved through phagocytosis by professional or amateur phagocytes. Failure in this process can lead to the development of inflammatory autoimmune or neurodegenerative diseases. AC clearance has been conserved throughout evolution, although many details in its mechanisms remain to be explored. It has been studied in the context of mammalian macrophages, and in the nematode *Caenorhabditis elegans*, which lacks "professional" phagocytes such as macrophages, but in which other cell types can engulf apoptotic corpses. In *Drosophila melanogaster*, ACs are engulfed by macrophages, glial, and epithelial cells. *Drosophila* macrophages perform similar functions to those of mammalian macrophages. They are professional phagocytes that participate in phagocytosis of ACs and pathogens. Study of AC clearance in *Drosophila* has identified some key elements, like the receptors Croquemort and Draper, promoting *Drosophila* as a suitable model to genetically dissect this process. In this review, we survey recent works of AC clearance pathways in *Drosophila*, and discuss the physiological outcomes and consequences of this process.

#### *Estee Kurant, University of Haifa, Israel*

*Edited by:*

*Reviewed by:*

*Yoshinobu Nakanishi, Kanazawa University, Japan Kimberly McCall, Boston University, United States*

#### *\*Correspondence:*

*Nathalie C. Franc<EMAIL_ADDRESS>Hui Xiao<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 18 October 2017 Accepted: 11 December 2017 Published: 20 December 2017*

#### *Citation:*

*Zheng Q, Ma A, Yuan L, Gao N, Feng Q, Franc NC and Xiao H (2017) Apoptotic Cell Clearance in Drosophila melanogaster. Front. Immunol. 8:1881. doi: 10.3389/fimmu.2017.01881*

Keywords: phagocytosis, apoptosis, macrophages, signaling pathways, *Drosophila melanogaster*

### INTRODUCTION

Programmed cell death is necessary for normal development and growth in multicellular organisms, which produce billions of apoptotic cells (ACs) daily (1, 2). Exogenous pathogenic microbes also threaten organisms' lives and development (3). Swift and efficient removal of ACs and pathogens is essential for maintaining tissue homeostasis. Failure in this process results in the release of potentially cytotoxic or antigenic molecules, causing inflammatory diseases or developmental autoimmune disorders (4–7). To clear ACs and pathogens, multicellular organisms have evolved a conserved cellular process named phagocytosis that is being carried out either by non-professional or professional phagocytes (8). The molecular mechanism of ACs clearance has been extensively studied in *Caenorhabditis elegans*, thus revealing relatively clear and detailed engulfment pathways (9). However, *C. elegans* lacks the professional phagocytes; instead ACs are engulfed by many neighboring cell types (10). Absence of a professional immune system in *C. elegans* may limit the extent to which these data can be applied to higher organisms. The fruitfly *Drosophila melanogaster* has also been used as a suitable model to study ACs clearance, in which ACs are engulfed by both nonprofessional phagocytes such as epithelial cells and professional phagocytes such as macrophages/ hemocytes and glial cells (11), providing the advantages for studying phagocytosis in mammals. ACs clearance proceeds when ACs expose "eat me" signals, which are recognized by phagocytes, thereby triggering signaling cascades that lead to internalization of the apoptotic corpse and its degradation by the phagocytic vacuole known as phagosome matures by fusing with lysosomes (12, 13). In this review, we will summarize the current research on phagocytosis of ACs in *D. melanogaster*, and which signaling pathways regulate this process, thereby giving a systematic and general overview of this process.

#### SIGNALING BY ACs IN PHAGOCYTOSIS

Apoptotic cells generated by programmed cell death or physical wounds are quickly and silently removed, to maintain tissue homeostasis or prevent auto-inflammatory responses (14, 15). Once cells begin to undergo apoptosis, cell death pathway is activated, and they release multiple signaling to recruit phagocytes, which contains three steps: the release of "find me" signals, the presentation of "eat me" signals, and the removal of "don't eat me" signals (16).

At the beginning of cell death, "Find me" signals are released from ACs to promote the migration of phagocytes to ACs. Lauber firstly identified lysophosphatidylcholine (LPC) as a "find me" signal, which is released from ACs in a caspase-3-dependent manner. They furthermore showed that the activation of calciumindependent phospholipase A2 by caspase cleavage contributed to the release of LPC (17). Two other molecules, sphingosine-1-phosphate produced by sphingosine kinase in a caspasedependent manner, and CX3CL1/fractalkine synthesized as a membrane-associated protein, have also been proposed to act as "find me" signals (18, 19). ATP and UTP that are released from ACs in a caspase-dependent manner have also recently been shown to act as "find me" signals for phagocytes (20). Whether these proposed "find me" signals are redundant or synergistic remains to be studied. Little evidence has shown that "find me" signals exist in *Drosophila*, but previous study revealed that H2O2 may be the immediate damage signal essential for the recruitment of hemocytes to wound regions in *Drosophila* embryos (21). Further research (22) found that Src42A–Draper–Shark signaling was important to recruitment of hemocytes by responding to wound-induced H2O2 in *Drosophila* embryos, which indicated that H2O2 may be *Drosophila* "find me" signal and Draper is responsible for the signal recognition. However, more evidence needs to be explored to verify this hypothesis.

"Don't eat me" signals (also known as self-associated molecular patterns) exist on healthy cells, playing inhibitory roles to prevent to be engulfed by phagocytes. Some examples of "don't eat me" signals include CD31, CD46, and CD47 in mammals (23).

"Eat me" signals are ligands, which can bind to engulfment receptors by moving to the surface of ACs. Engulfment receptors recognize and bind either directly to the apoptotic "eat me" signal, or through bridging molecules that bind the "eat me" signal. The best-studied and evolutionarily conserved "eat me" signal reported in human, *Drosophila*, and *C. elegans* is phosphatidylserine (PS) (24), a phospholipid exposed on the surface of ACs (25, 26). PS is a plasma membrane (PM) aminophospholipid maintained on the inner leaflet of live cells through aminophospholipid translocase activity (27, 28). After cell induced by apoptosis, aminophospholipid translocase is inactivated while a scramblase is activated to induce PS exposed to the cell surface in an ATPindependent manner (28). A recent study has shown that ACs can generate molecular memory in macrophages, priming them to recognize tissue wounds or microbes (29). This subsequently causes macrophages to produce pro-inflammatory signals and boost the innate response at sites associated with extensive AC death in *Drosophila* (29).

#### ENGULFMENT RECEPTORS AND RELATED SIGNAL PATHWAYS

In *Drosophila*, there are three cell types reported to function as phagocytic cells: professional phagocytes—macrophages/hemocytes, glial cells, and non-professional phagocytes—epithelial cells (30–32). Hemocytes are macrophage-like cells reported to engulf ACs or dendrite debris during pruning of *Drosophila* sensory dendrites (33) and embryogenesis (34). *Drosophila* glia act much similar role in engulfing dying cells or degenerating axons of the nervous system as their counterparts in mammals (35), degenerating dendrites are primarily cleared by the epidermal epithelia (36).

"Eat me" signals secreted by ACs are recognized by engulfment receptors, which are specifically expressed on the surface of phagocytic cells. In *C. elegans*, two seemingly independent engulfment signaling pathways have been genetically identified, which share similar functions both in fly and mouse, indicating that the process of ACs clearance is evolutionarily conserved. CED-1, a conserved transmembrane receptor protein, Draper in fly, MEGF10 in mouse, which have similar function in recognizing ACs, transducts the phagocytotic signal through its adaptor protein CED-6 (dCed-6 in fly, GULP in mouse) to regulate downstream effectors (37, 38). The CED-2, -5, -10, and -12 signaling pathway is believed to act downstream of the PS receptor PSR-1, a *C. elegans* homolog of mammalian PSR (39), which relates to ACs cytoskeletal rearrangements. Some of the abovementioned genes possess *Drosophila* counterparts, suggesting that fruitfly phagocytes share similar pathways to engulf ACs. Meanwhile, *Drosophila* has its own engulfment receptor, yet a more detailed mechanism remains to be unveiled in *Drosophila*.

#### Croquemort

In 1996, Franc and colleagues cloned the first *Drosophila* engulfment receptor on embryonic macrophages, Croquemort (Crq), which shares 23% identity with human CD36. In mammals, CD36 act as a scavenger receptor engulfing ACs (40) and regulates the host inflammatory responses (41, 42). Crq expresses specifically on *Drosophila* plasmatocytes, which become macrophages as they encounter ACs from late stage 11 of embryogenesis (43). Using AC-labeling and Crq immunostaining experiments, Crq was shown to be required for efficient phagocytosis of ACs, which was also confirmed *in vivo* (34). Crq is structurally unrelated to either CED-1 or PSR-1 (34), and how it promotes phagocytosis, including the identity of its ligand, is still unknown (44).

In addition to macrophages clearing ACs during embryogenesis, epithelial cells are responsible for prompt clearance of degenerating neurites to maintain tissue homeostasis and prevent inflammatory responses during development (36). Knocking out *crq* results in AC clearance defects by macrophages; however, it has no effect on engulfment of dendrites in epithelia. Further studies showed that *crq* was required for phagosome maturation during this process, while loss-of-function of *crq* leads to homotypic phagosome fusion defect, though it is not necessary for phagosomes to progress through the Rab7+ positive stage (43). Besides, recent research revealed that *crq* mutant flies are susceptible to environmental microbes and infection, and that Crq is required for engulfment of bacteria in parallel to the Toll and Imd pathways, which play key roles in the innate immune system (45).

#### Draper

Freeman and colleagues first identified the homolog of CED-1 in *Drosophila*, named Draper (Drpr), which strongly expressed on glial and macrophage membranes, and found that it was required for the engulfment of apoptotic neurons and for larval locomotion (35). Similar to CED-1 in nematode and MEGF10 in human, Drpr encodes 15 extracellular atypical EGF repeats, a single transmembrane domain, and a novel intracellular domain (35). Manaka and colleagues confirmed the role of Drpr in glia and hemocytes/ macrophages, showing that it plays a role in the phagocytosis of ACs (44), suggesting that the Drpr pathway plays similar role in *Drosophila* as the ced-1/6/7 pathway in *C. elegans*. Glial cells expressing Drpr are essential for the pruning of *Drosophila* mushroom body γ neurons, Awasaki et al. detected that *Drosophila ced-6* (mouse *gulp*) expressed in the same glial cells as *drpr* (46), genetic evidence showed that *drpr* and *ced-6* played role in engulfing γ neuron axon in the same pathway, meanwhile, the experiment *in vitro* confirmed that Ced-6 N-terminal might interact with the intracellular region of Drpr (47). Different from *C. elegans* Ced-7, an ABC transporter, which both expresses in ACs and engulfment cells for efficient phagocytosis, the homolog in *Drosophila*, has not yet been studied. *Drosophila* Shark, a non-receptor tyrosine kinase also plays an important role in removing cell corpses or debris mediated by Drpr through binding to its intracellular domain (48). The Src family kinase Src42A phosphorylates Drpr to allow its intracellular domain to interact with Dmel/Ced-6, thus activating the Drpr pathway and promoting phagocytosis of pruned axons and degenerating neurons by glial cells (47).

In addition to the Ced-1, -6, and -7 signaling pathway, Ced-2, -5, -10, and -12 were found to act in a parallel and yet partially redundant pathway that controls actin cytoskeleton rearrangement in cell corpse engulfment and cell migration (49). For *Drosophila*, although the homologs of CED-2, -5, and -10 correspond to CG1587, myoblast city, and Rac2, respectively, their function in ACs clearance has not been deeply studied. The *Drosophila* homolog of Ced-12, Dmel/ced-12, was found to be required for cell clearance in macrophages, function in a genetically distinct pathway compared with Drpr, which further indicated that the phagocytosis signal pathways are evolutionary conserved (50).

#### Integrin

Integrins are conserved heterodimeric transmembrane receptors, forming by two subunits called α and β (51, 52). The involvement of integrins in phagocytosis of ACs was first described in mammals (53). Ina-1, an α subunit of *C. elegans* integrin, was also reported to participate in cell corpse removal (54). In *Drosophila*, there are five α- and two β-subunits. Nagaosa and colleagues found that loss-of-function of *Drosophila* integrin *βv* results in reduced levels of AC clearance, while reexpressing *βv* in integrin *βv*-lacking fly hemocytes rescues their phagocytosis-defective phenotype (55). Flies lacking either integrin *βv* or Drpr showed almost the same level of phagocytosis, while loss of these two receptors further decreased phagocytosis, which indicated that integrin *βv* and Drpr act independently. As Drpr was shown to act upstream of CED-6 and CED-10, the integrin *βv* appears to act upstream of the other engulfment pathway CED-2–CED-5– CED-12. However, Crk and Mbc, the *Drosophila* homologs of *C. elegans* CED-2 and CED-5 have not been observed to participate in the phagocytosis of ACs at least by embryonic hemocytes, thus the molecular signaling downstream of *βv* remains unknown (55). Further research indicated that *Drosophila βv* acts as a phagocytic receptor to also promote clearance of *Staphylococcus aureus via* peptidoglycan binding on this bacterium (56). Another *Drosophila* integrin α-subunit, αPS3, also cooperates with *βv* in hemocytes and serves as an engulfment receptor for phagocytosis of ACs and *S. aureus* (57). In *Drosophila* ovary, highly polarized epithelial follicle cells (FCs) can engulf germline debris *via* their apical side. Meehan et al. (58) found that integrin heterodimer αPS3/βPS were apically enriched in engulfing FCs, which are required for engulfment of ACs by FCs. Thus, integrins are evolutionally conserved receptors that participate in AC clearance.

# BRIDGING MOLECULE

Several engulfment receptors have been identified that mediate phagocytosis of ACs, yet little is known about their precise mechanism of action, or whether they cooperate or act alone. Several molecules have been characterized that function upstream of Drpr to recognize ACs that are considered as "bridging molecules."

### Six-Microns-Under (Simu)

Kurant and colleagues characterized a transmembrane protein named Simu, which is highly expressed on the surface of glial cells in the nervous system and macrophages elsewhere (59). Simu acts upstream of Drpr promote the recognition and engulfment of ACs (59). It strongly binds to ACs, through its EMILIN-like domain without membrane anchoring. Furthermore, Kurant and colleagues demonstrated that SIMU recognizes and binds PS secreted on ACs through its N-terminal EMILIN (EMI)-like domain, while the C-terminal NIM3 and NIM4 repeats regulate Simu affinity to PS (60). In addition, caspase activity is required for clearance of ACs by glial cells (60). However, the interaction mechanism between Simu and Drpr during clearance of ACs remains unclear, as Kurant and colleagues were failed to detect a directly physical linkage between Simu and Drpr (59). Thus, it seems likely that other molecules are required to connect these proteins (61).

### Calreticulin (Calr), Pretaporter (Prtp), and *Drosophila* Calcium-Binding Protein 1 (DmCaBP1)

Various proteins and lipids from the endoplasmic reticulum (ER) have also been found to be exposed at the surface of human ACs (62). Nakanishi and colleagues identified three ER proteins acting upstream of Drpr to promote phagocytosis in *Drosophila* (63–65). They showed that *Drosophila* Calr existed at the surface of living cells and reassigned to form aggregates upon apoptosis without change of the amount and expression at the cell surface; and that in a *Drosophila* mutant strain with reduced level of Calr, the level of phagocytosis of ACs was about a half of that observed in wild-type embryos (63). Thus, like PS, Calr is considered as a marker for phagocytosis of ACs in *Drosophila*. Through protein pull-down analysis, Nakanishi isolated an ER protein binding to the extracellular region of Drpr, with a signal peptide at the N-terminal and an ER retention motif at the C terminal, named Prtp. They found that Prpt relocated from ER to cell surface during apoptosis in *Drosophila* S2 cells (64), and they further showed that loss-of-function of *prtp* leads to reduced level of AC clearance both by embryonic hemocytes and embryonic glia. Reexpression of *prtp* in hemocytes did not rescue this defect while the ubiquitous expression did, which indicated that Prtp functions in ACs to promote phagocytes' engulfment (64). The DmCaBP1 is released and externalized from ACs, to bind to the extracellular region of Drpr (65). Loss of either *prtp* or *DmCaBP1* led to a reduced level of AC clearance in *Drosophila* embryos, but the double mutant did not cause a further decreased in phagocytosis, which indicated that they act in the same pathway. As apoptosis induced, DmCaBP1 is externalized from ACs and serves as a bridging molecule to connect ACs and phagocytes, promoting efficient and timely phagocytosis to occur.

### E3, UBIQUITIN PROTEASOME PATHWAY

By screening for genes required for efficient phagocytosis of ACs in *Drosophila* macrophages *in vivo*, Silva and colleagues identified *pallbearer* (*pall*), which encodes an F-box protein (66). F-box proteins are generally part of Skp/Cullin/F-box (SCF) complexes that act as E3 ligases targeting phosphorylated proteins to ubiquitylation and degradation *via* the 26S proteasome (67). In addition to F-box protein, the SCF complexes contain three constant polypeptides—Skp1, Cullin1 (Cul1), and Rbx1, which have their counterparts in *Drosophila*. In *Drosophila*, six Skp proteins have been identified; and only SkpA strongly expressed in the embryos (68), and Bocca reported that SkpA and Rbx1 interact with Lin19 (dCul1) respectively (69). Silva and colleagues showed that Pall physically interacts with SkpA *via* its F-box domain, the loss function of either Lin19 or SkpA resulted in phagocytosis-defective phenotype, which indicated that they constitute complexes to promote phagocytosis of ACs (66). Xiao and colleagues then identified one substrate of the Pall–SCF complex, namely, the ribosomal protein S6 (Rps6) (70). The F-box protein Pall interacts with phosphorylated Rps6, which induces its ubiquitination and degradation *via* the 26S proteasome pathway (70). As a consequence, Xiao and colleagues further showed that the Rac2 small GTPase was upregulated and activated, triggering actin cytoskeleton rearrangement and thus promoting the clearance of ACs (70). They also showed that Pall translocates from the nucleus to the cytoplasm upon AC exposure (70). However, the AC signal and molecular pathway that leads to Pall nuclear export has not yet been identified. Furthermore, the nature of the kinase that phosphorylates Rps6 upstream of its physical interaction with Pall and how the degradation of phosphorylated RpS6 results in higher levels and activation of Rac2 remain to be deciphered.

### CALCIUM SIGNALING

Calcium signaling is a second messenger, which participates in a number of cellular processes (71). Studies have identified several Ca2+ signaling genes that are required for AC removal in *Drosophila*. Cuttell and colleagues identified Undertaker (Uta) (also known as retinophilin), a *Drosophila* protein with membrane occupational recognition nexus repeats related to Junctophilinlike proteins, as required for Drpr-mediated phagocytosis (72). Junctophilins form junctional complexes between the PM and the ER or sarcoplasmic reticulum (SR) Ca2+ storage compartments that allow for cross talk between Ca2+ channels at the PM and the ER/SR Ca2+ channels (73). Cuttell and colleagues showed that the *Drosophila* ryanodine receptor, Rya-r44F, a Ca2+ channel on the ER membrane, also plays role in phagocytosis of ACs mediated by the Drpr pathway (71). They found that *uta* genetically interacts with *rya-r44F* upstream of the Drpr and Dmel/Ced-6 pathway to activate their downstream signaling cascade for efficient phagocytosis of ACs (72). Thus presumably, Uta forms junctional complexes between the PM and the ER to trigger the release of Ca2+ from the ER/SR compartment *via* Rya-R44F. Conversely, they showed that *drpr* and *Dmel/ced-6* are required for store-operated calcium entry (SOCE) *via* Stim and Orai (71). Thus, signaling downstream of Drpr and Dmel/Ced-6 may promote and/or maintain Uta-mediated junctional complexes, consequently mediating ER Ca2+ release to SOCE *via* Stim and Orai. It appears that Ca2+ functions in Drpr signaling downstream during both recognition and internalization of ACs, and Uta plays a central role both in Ca2+ homeostasis and phagocytosis. A similar link between Ca2+ homeostasis and AC clearance has been found in mammalian systems and *C. elegans* (74). Interestingly, a novel mechanism has been found by Weavers that *Drosophila* embryonic macrophages generate a memory after the uptake of ACs, priming them to detect tissue damage or infections. Engulfment of ACs associates with calcium bursts, increasing Drpr expression, which is important for the macrophages to rapidly respond and migrate to subsequent injury or infections (29).

#### CROSS TALK WITH INNATE IMMUNE RESPONSE

As phagocytosis is crucial for the normal development, it also plays important role in the immune response for the removal of ACs and pathogens (3, 24). The mechanisms that mediate phagocytosis of bacteria and how it interacts with other innate immune responses defense remain elusive. Hashimoto and colleagues showed that Drpr promotes phagocytosis of *S. aureus*, and *drpr* mutant flies show reduced resistance to a septic infection with *S. aureus* (75). *ltaS* encodes an enzyme responsible for the synthesis of lipoteichoic acid in *S. aureus* that acts as a ligand for Drpr in phagocytosis of *S. aureus* by *Drosophila* hemocytes. The integrin *βv* subunit promotes phagocytosis of *S. aureus* by

Figure 1 | Overview of corpse recognition and disposal in *Drosophila*. Key components positioned according to their roles in corpse recognition, internalization, and processing. Some important questions are also indicated. Three engulfment receptors located at the plasma membrane. CRQ (Croquemort), a membrane two-path protein, expressed mainly in the macrophages and its ligand and downstream have not studied clearly; Draper (Ced-1), a membrane single-path protein, expressed mainly in glia of CNS and hemocytes, with its ligands are shown in this figure, phosphorylated Draper interact with Ced-6 thus elevates Jun N-terminal kinase (JNK) signal and maintain Ca+ homeostasis; JNK promotes Drpr enrichment both in glia and follicular epithelia (under Ced-12 activation). Integrins, a heterodimer protein, function in epithelial follicle cells and hemocytes to engulf cell corpses. The solid arrows mean the relationship between two proteins; the dotted arrows mean the molecular mechanism linking two pathways or proteins unknown. The purple circles with "P" in them mean phosphate groups. The orange circles with "AC" in them mean apoptotic cells; the rectangle outlined by blue lines means engulfment cells; different proteins or molecules are represented by colored shapes as shown in this figure.

binding to peptidoglycan of this bacterium (56), and the integrin αPS3 subunit cooperates with *βv* in this process (57). Guillou and colleagues showed that *crq* defective mutant flies appeared to be more susceptible to environmental microbes both during development and at adulthood, they further demonstrated that *crq* is required for microbial phagocytosis (45). Interestingly, AC clearance by *Drosophila* macrophages appears essential in priming these cells to respond to subsequent microbial infections *in vivo* (29). Macrophages that have not engulfed ACs fail to take up *E. coli*, while those that have previously engulfed ACs can recognize and take up *E. coli*, ultimately mediating the bacterium phagosomal degradation (29).

### JUN N-TERMINAL KINASE (JNK) PATHWAY

After recognition of ACs by macrophages and epithelial cells in mammals, the stress-activated MAP kinases JNK and p38 are activated at the early stage (76, 77). In *Drosophila* imaginal epithelia, normal imaginal cells exert an antitumor effect as oncogenic cells emerged to eliminate them (78). Ohsawa et al. revealed that the antitumor effect from surrounding cells was mediated by the activated JNK signaling, thus promoting the elimination of premalignant neighbors by engulfment (79). In *Drosophila* ovary, dying germline cells are cleared by neighbor follicular epithelia, which required Drpr signal pathway and activated JNK signal (80). During this process, Drpr acts upstream to activate JNK pathway, but another regulator exists to activate JNK pathway, which has not been studied. Their results suggested that the dying germline activates Drpr–JNK pathway, then JNK activity feeds back to increase Drpr expression in engulfing cells, which seem to be a circuit. Interestingly, although Ced-12 was showed to promote AC clearance in an independent pathway compared with Drpr, in *Drosophila* ovary, Timmons et al. (81) found that Ced-12 act upstream of JNK, which can to increase Drpr expression, similar to described earlier in *Drosophila* glia. As mentioned previously, glial cells play an important role in removing ACs during *Drosophila* embryonic development, neuronal pruning, and axonal degeneration (47, 59). Shklover showed that excess activation of JNK signaling in *Drosophila* embryonic glial cells does not affect the levels of Simu and Drpr expression but still promotes their apoptotic death and upregulates their phagocytic capacity by glial cells (82). As mentioned earlier, JNK signaling in follicular epithelia upregulates expression of Drpr, indicating that the phagocytosis induced by JNK signal may be tissue-specific. Recently, research showed that *Drosophila* glia upregulate their basal ability after neuronal injury, to phagocytosis through activation of the JNK pathway, which leads to the elevation of DRPR level (80, 83).

As mentioned previously, Weavers and colleagues proposed that ACs generate a molecular memory within macrophages, priming them to repair tissue damage and fight infection (29). They showed that JNK signaling is essential for macrophage detection of tissue damage and bacteria, as the uptake of ACs triggers calcium bursts in macrophages that induce JNK activation and signaling, ultimately leading to Drpr upregulation of expression (29).

#### CONCLUSION

Efficient and proper corpse clearance is important to maintain normal growth and prevent inappropriate inflammatory response, defective clearance of ACs often bring forth various diseases, such as autoimmune diseases, neurodegeneration, atherosclerosis, and Alzheimer's disease*.* As AC clearance pathways were conserved from invertebrate to mammals, the typical pathways—*ced-1*, *-6*, *-7*, and *ced-2*, *-5*, *-10*, and *-12*, most-studied in worms or in mammals, also exist in flies. Over the past few decades, researchers have unveiled some of the molecular mechanisms of AC clearance in *Drosophila*. The process is outlined in **Figure 1**. However, multiple important questions concerning clearance mechanisms remain to be answered, and more detailed mechanisms remain to be explored. How does Crq recognize ACs and what is the ligand of Crq? Are there other engulfment receptors or regulators required for phagocytosis of ACs in *Drosophila*? How is Crq expression regulated by ACs? How does the translocation of Pall from nucleus to cytoplasm happen and which pathway regulates

#### REFERENCES

this event? Are the same regulatory mechanisms involved? What are the molecular mechanisms that mediate phagocytosis of bacteria and how do they overlap or differ from that of ACs? How does phagocytosis interact with other innate immune responses defense? To answer these questions, further studies of engulfment signals and the phagocytic machinery is required. In conclusion, our understanding of AC engulfment mechanism in *Drosophila* will enhance our theoretical foundation in this area, and provide a powerful complement to the research in mammals that could be useful for the development of therapeutic strategies to control diseases related to defective cell clearance.

#### AUTHOR CONTRIBUTIONS

QZ, AM, LY, NG, QF, NF, and HX wrote and reviewed the manuscript.

#### FUNDING

This work was partially supported by the National Natural Science Foundation of China (Grant No. 31671439), Natural Science Foundation of Shaanxi Province, China (Grant No. 2017JM3008), the program of Innovative Research Team for the Central Universities (Grant No. GK201701005), the Fundamental Research Funds for the Central Universities (Grant No. 2017TS034), the Innovation Fund for undergraduate students (Grant No. KY2017ZD018), and Thousand Youth Talents Plan.

clearance of DmCaBP1, an endoplasmic reticulum protein of *Drosophila*. *J Biol Chem* (2012) 287:3138–46. doi:10.1074/jbc.M111.277921

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer KM declared a past coauthorship with the author NF to the handling editor.

*Copyright © 2017 Zheng, Ma, Yuan, Gao, Feng, Franc and Xiao. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Clearance of Apoptotic Cells by Tissue epithelia: A Putative Role for Hepatocytes in Liver efferocytosis

*Scott P. Davies1 , Gary M. Reynolds1,2,3 and Zania Stamataki1 \**

*1Centre for Liver Research, College of Medical and Dental Sciences, Institute for Immunology and Immunotherapy, University of Birmingham, Birmingham, United Kingdom, 2 Centre for Liver Research and National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit, College of Medical and Dental Sciences, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, United Kingdom, 3University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom*

Toxic substances and microbial or food-derived antigens continuously challenge the liver, which is tasked with their safe neutralization. This vital organ is also important for the removal of apoptotic immune cells during inflammation and has been previously described as a "graveyard" for dying lymphocytes. The clearance of apoptotic and necrotic cells is known as efferocytosis and is a critical liver function to maintain tissue homeostasis. Much of the research into this form of immunological control has focused on Kupffer cells, the liver-resident macrophages. However, hepatocytes (and other liver resident cells) are competent efferocytes and comprise 80% of the liver mass. Little is known regarding the mechanisms of apoptotic and necrotic cell capture by epithelia, which lack key receptors that mediate phagocytosis in macrophages. Herein, we discuss recent developments that increased our understanding of efferocytosis in tissues, with a special focus on the liver parenchyma. We discuss the impact of efferocytosis in health and in inflammation, highlighting the role of phagocytic epithelia.

#### *Edited by:*

*Amiram Ariel, University of Haifa, Israel*

#### *Reviewed by:*

*Christina Janko, Universitätsklinikum Erlangen, Germany Joan Clària, Hospital Clinic-Barcelona University School of Medicine, Spain*

#### *\*Correspondence:*

*Zania Stamataki<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 16 October 2017 Accepted: 08 January 2018 Published: 25 January 2018*

#### *Citation:*

*Davies SP, Reynolds GM and Stamataki Z (2018) Clearance of Apoptotic Cells by Tissue Epithelia: A Putative Role for Hepatocytes in Liver Efferocytosis. Front. Immunol. 9:44. doi: 10.3389/fimmu.2018.00044*

Keywords: efferocytosis, phagocytosis, liver, hepatocytes, regeneration, apoptosis, necrosis, cell death

# KEY POINTS

# EFFEROCYTOSIS IN THE LIVER

Efferocytosis, the clearance of dead and dying cells, is important to prevent tissue damage and promote the resolution of inflammation (1). The liver has evolved into an expert in defusing biochemical threats emanating from food or microbial antigens, which reach the organ along with 75% of its blood supply that arrives through venous blood from the gut. Hepatocytes comprise 80% of liver cells and constitute the biochemical powerhouses of the liver parenchyma, and as a result they often perish in their duties to absorb toxic substances. To cope with loss of hepatic epithelia, the liver has evolved the remarkable ability to regenerate.

To perform their detoxification roles, hepatocytes are strategically organized roughly into two hepatocyte-thick cords, flanked by a thin layer of fenestrated endothelia (**Figure 1**). Nutrient-rich blood enters the liver *via* the portal vein and oxygen-rich blood *via* the hepatic artery, which, together with a bile duct, form the liver portal triad (**Figure 1A**). Blood from both sources mixes in the specialized hepatic capillaries termed sinusoids, and drains toward the central vein. Hepatocytes near the portal triads (designated zone 1) can be damaged by the inflammatory infiltrate during interface hepatitis, when immune cells cross the sinusoidal endothelia and reach the parenchyma. Zone 2 is found mid-distance from a portal triad and the draining central vein (zone 3). Periportal hepatocytes

Figure 1 | Organization of liver-resident and recirculating efferocytes. (A) Hepatocytes are spread over three zones, exposed to different levels of oxygen and nutrients. Hepatocytes in zone 1 proximal to the portal triad (portal vein, hepatic artery, bile duct) have access to arterial and venous blood entering the liver through the circulation. Hepatocytes in zone 3 have less access to oxygen and nutrients and are exposed to blood draining into the central vein. Hematoxylin-eosin stain, scale bar represents 50 µm. (B) A plethora of liver resident and recirculating cells are able to engulf apoptotic and necrotic cells and clear them to maintain tissue homeostasis. Kupffer cells, monocytes and macrophages (mϕ) are the best-characterized efferocytes in the liver.

near zone 1 have access to oxygenated blood from the hepatic artery, and nutrients from the portal blood supply that arrives from the gut. Oxygen and nutrient levels reduce toward the central vein and hepatocytes in zone 3 are found in hypoxic conditions. Fenestrations in the sinusoids allow hepatocytes access to solutes and immune cells reaching through the fenestrations from the circulation (2, 3), but prevent unregulated migration of immune cells to the parenchyma (4).

Cells that perish in the sinusoidal spaces are cleared by circulating phagocytes (monocytes, dendritic cells, neutrophils), liver-resident macrophages termed Kupffer cells, and by sinusoidal endothelia (**Figure 1B**). The best-characterized liver efferocytes are macrophages, both those derived from monocytes infiltrating from the circulation, and the self-renewing populations of Kupffer cells. It is understood that professional phagocytes are activated during injury and adapt their phenotype following the encounter of cellular debris, danger signals, and soluble mediators of the inflammatory milieu. The critical role of liver macrophages including Kupffer cells in the ebb and flow of inflammation was recently reviewed by Tacke's group (5, 6).

Activated hepatic stellate cells can also engulf apoptotic hepatocytes, which in turn lead to increases in tumor growth factor-β (TGF-β) secretion (7). Biliary epithelial cells (BECs) also take part in efferocytosis of neighboring apoptotic cells; an important adaptation for diseases associated with increased BEC apoptosis such as primary biliary cholangitis (8). The phagocytic activity of hepatocytes was noted in 1992 (9). Hepatocyte efferocytosis assists in parenchymal housekeeping to rapidly dispose of cell remnants and prevent excessive inflammation.

Hepatocyte death from biochemical toxicity (*necrosis*) occurs in health as part of normal homeostasis, however, liver damage is exacerbated in infection or in alcoholic, drug or ischemia-induced liver injury where large areas of necrotic lesions are evident (**Figure 2**). Acute-on-chronic liver failure is a syndrome associated with exacerbation of hepatitis B infection (HBV) and characterized by broad areas of hepatic necrosis in cirrhotic patients (**Figure 2A**). Lymphocyte infiltration is often seen in the parenchyma in chronic liver diseases. Crispe and others have elegantly put forward the "graveyard theory" where the liver is primary site for the disposal of spent immune cells (10). **Figure 2B** shows hepatic epithelia in the process of engulfing immune cells that have perished in the parenchyma, and this is seen predominantly near the portal regions. Conversely, in cases of acute liver injury such as paracetamol overdose (POD), hepatocyte necrosis due to loss of ATP is noted around the centrilobular regions (zone 3, **Figure 2C**). Histological features of necrotic hepatocytesinclude eosinophilic degradation and pyknotic nuclei, which are readily detectable by hematoxylin-eosin staining (inset, **Figure 2C**).

Hepatocytes also clear away cells that have triggered the molecular cascade of events of programmed cell death (*apoptosis*) (**Figures 2B,D**), but can actively destroy live autoreactive immune cells by direct engulfment as noted for CD8+ T cells undergoing *suicidal emperipolesis* (11). Immune cell death and liver damage are exacerbated in chronic liver inflammation of multiple

etiologies, including autoimmune, metabolic, viral, and genetic diseases (12, 13). The rapid processing of dead and dying cells is vital to moderate inflammation (12, 14, 15).

It is remarkable how little we know about the molecular mechanisms that govern the ability of the largest internal organ in the body to mediate the clearance of damaged or dying cells, given that this is one of the liver's major functions. Herein, we bring together research on hepatocyte efferocytosis and place it into context with current molecular knowledge on the clearance of dead cells by immune phagocytes.

## CLEARANCE OF APOPTOTIC AND NECROTIC CELLS

Cells die through a wide array of processes, each situational and requiring their own dedicated cascade of signaling events. The most frequent forms of cell death are attributed to apoptosis or necrosis. Apoptosis, an active form of programmed cell death, is characterized by the initiation of specific inducible pathways (16, 17). This includes the extrinsic pathway; the engagement of extracellular signals, including Fas ligand (FasL) (18) and tumor necrosis factor family cytokines (TNF) (19), amongst others, to their respective death receptors which initiates intracellular death signaling. Apoptosis can also be triggered intrinsically; certain signals, such as a lack of growth factors, endoplasmic reticulum stress or DNA damage, can induce a shift in expression of Bcl-2 family mitochondrial proteins (20). Increased activity of proapoptotic proteins lead to cytochrome C release and caspase 9 activation. Apoptosis pathways result in the activation of effector caspases (3, 6, and 7), which in turn begin to proteolytically degrade the cell's components. Apoptotic cells are generally smaller than live cells and can be identified by the formation of surface blebs (16).

Necrosis is considered a passive, unprogrammed type of cell death and is often incurred accidentally, although active mechanisms of necrosis have also been reported (21). While multiple mechanisms can induce necrosis, the major causes are attributed to compromising of the plasma membrane, or depletion of energy (22). Furthermore, apoptotic cells can be converted to necrotic cells (also known as *secondary necrosis*) if ATP levels fall below the quantity required to complete the active apoptotic process (23). The appearance of necrotic cells is often swollen with disrupted organelle and plasma membranes (24). The nucleus is often broken down and will be unstained by hematoxylin (**Figure 2**). As necrosis often occurs in areas of tissues, rather than the single cell death hallmark of apoptosis, often multiple necrotic cells can be identified in one area. Due to their lack of integrity, necrotic cells will often form cell debris, which can induce liver damage if not cleared swiftly, as we discuss in later sections.

Upon the death of a cell, its corpse must be cleared through efferocytosis. This is a specialist form of phagocytosis, whereby fragments of the dying cells are engulfed by other cells, which in turn degrade and recycle their components. Although both apoptotic and necrotic cells are often captured by the same efferocytes, each are recognized through different means and yield differing response in the predatory cell (25). Apoptotic cells are most commonly recognized through the display of the phospholipid phosphatidylserine (PtdSer) on the outer leaf of the plasma membrane that can be recognized by many receptors [phosphatidylserine receptors (PSRs)] directly (26) or *via* association with low-density lipoprotein (27, 28). Of note, in a rat liver model, it was shown that recognition of apoptotic cells from mice or humans was reduced compared to rat cells; it is therefore possible that species-specific recognition molecules can mediate efferocytosis (29).

A phenotypic aspect of apoptotic cells is that, although shriveled, the cell remains intact as a singular body. This allows for a clean removal of the dying cells by efferocytes, usually without provoking an inflammatory response. How intact a necrotic cell remains is reflected through the manner in which cell death was induced. As such, multiple modalities for necrotic cell recognition are necessary to guarantee their clearance. Some reports have suggested that necrotic cells can also be recognized by PSRs (30). However, due to the lack of integrity of most necrotic cells, they are often recognized through molecules exposed by necrotic death (25). The same mechanisms are also used to detect pathogens. For example, complement receptors and Fc receptors detect opsonized necrotic cells, and this recognition can trigger signaling events that activate the phagocyte (31–35). As such, necrotic cells are engulfed through the detection of autoantigens, which often increases the risk for autoimmune disease. Necrotic cells can also be indirectly recognized through opsonin engagement of other cellular components. For example, ficolin-2 and -3 have been shown to bind DNA, facilitating the clearance of late-apoptotic/necrotic cells through interactions with calreticulin (33, 36). A ubiquitous mechanism for clearance of necrotic cells remains uncertain.

The differences between recognition, and thus further downstream signaling of apoptotic and necrotic cells, result in conversing consequences for the efferocyte (37). Apoptotic cell clearance generally leads to the production of anti-inflammatory stimuli and pro-resolution signals for inflammation such as interleukin 10 (IL-10) and TGF-β (38). Conversely, necrotic clearance generally results in pro-inflammatory signaling, as

many of the recognition receptors are also required for pathogen recognition. In the liver, the signals associated with hepatocyte death were recently reviewed by Brenner and colleagues (39). In this work the importance of the extent and duration of dead cell accumulation was highlighted, as mild and localized cell death can aid regeneration and exert hepatoprotective effects. Equally, prolonged and wide-spread cell death can exacerbate liver injury.

### EXPERIMENTAL SYSTEMS TO STUDY EFFEROCYTOSIS

Multiple techniques have been described for both *in vivo* and *in vitro* studies of efferocytosis. Fluorescent dye-labeled efferocytes can be "fed" alternatively labeled dead cells under varying conditions and time courses. Early apoptosis can be confirmed by Annexin V labeling of the cell surface as it binds directly to PtdSer, although care must be taken when studying certain activated cell types or using calcium-sensitive protocols (40). Later stages of apoptosis or necrosis are often confirmed with cell impermeable DNA dyes such as 7AAD or TOPRO-3 iodide, which can enter cells once the membrane is compromised. Combined labeling with Annexin V and a membraneimpermeable DNA label was developed to identify the stages of apoptotic cells in more detail (41). Cells can then be assessed by flow cytometry, or imaged by fluorescent microscopy. Complete internalization of dead cells can be confirmed by lack of access to membrane dyes added to the culture media (such as CellMask Plasma Membrane Stains, Thermo Fisher Scientific) or demonstration of efferosome acidification using pH indicator dyes (**Figure 3**). Quantitative analyses by confocal and timelapse microscopy can be useful to determine the frequency and kinetic of efferocytosis *in vitro*.

Figure 3 | Visualizing efferocytosis by confocal microscopy. Hepatic epithelia were cocultured with violet-labeled staurosporin-treated apoptotic Jurkat T cells in the presence of pHrodo red, which only fluoresces in conditions of low pH (Thermo Fisher Scientific). CellMask Plasma Membrane stain was added to the culture media to label all exposed cell membranes before imaging. (A) Non-internalized apoptotic cells (blue) attached to hepatocytes were labeled by CellMask Plasma Membrane in white, and they were not labeled by pHrodo red dye (white arrow). (B) Internalized dead cells were not accessible to the membrane dye, confirming internalization (yellow arrow). Complete internalization into an acidic compartment was confirmed by pHrodo red, which detected efferosome acidification as early as 3 hours following engulfment. The scale bar indicates 5 µm.

Fluorescent labeling of dead cells and efferocytes may also be adapted for flow cytometry-based studies, whereby doublepositive cells represent efferocytes containing cargo. This form of analysis has been used to study the clearance of neuraminidasetreated red blood cells in mice (42). Other studies opt to analyze efferocytosis using downstream secreted molecules as proxy to utilize alternative techniquessuch asreporter assays.The capacity of Scavenger Receptor Class F Member 1 (SCARF1) to act as a dead-cellreceptor ontransfectedHEK293T cells,for example,was confirmed using IL-8 mRNA production as a marker of NF-κB activation following apoptotic and necrotic cell efferocytosis(43).

Efferocytosis is not often as straightforward to detect *in vivo*. Fluorescent labeling can enable temporal measurements in mouse models by intravital imaging of the liver (44), but the technique remains to be adapted successfully for use in human tissues *ex vivo*. Molecular markers of cell death for use with fixed tissue are often important for the confirmation of efferocytosis. Caspase 3/7 activation or their effects can be measured to delineate apoptotic bodies by immunohistochemistry (IHC) or immunofluorescence (IF) (45). DNA end-labeling is frequently used to confirm the death of cells in tissues. End-labeling involves the addition of labeled nucleotides to DNA breaks induced throughout multiple modalities of death, using a DNA polymerase. This was historically used for *in situ* end labeling (ISEL) of fixed tissue sections as part of IHC chromagen staining (**Figure 2D**) (46). This was then adapted for the creation of terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) (47) which substitutes a polymerase for TdT. This adaptation allows for the use of many different modified forms of labeled nucleotides (often dUTP), such as non-reactive protein tags or fluorophores. TUNEL staining has been altered to specifically identify cells in late-stage apoptosis.

Further stains for cell membrane proteins or specific nucleic proteins can be used to determine complete engulfment of dead cells. Many of these techniques were exemplified in a recent study of macrophage/monocyte efferocytosis in models of acute liver injury (48). In this work, Antoniades and colleagues studied the mechanism of resolution of liver inflammation through apoptotic cell clearance by macrophages/monocytes *via* Mertyrosine kinase receptor (MerTK). Staining for myeloperoxidase (activated neutrophils) combined with TUNEL allowed for the identification of apoptotic neutrophils in human liver, both through IHC and IF staining. Additionally, fluorescent monocytes were cocultured *in vitro* with alternatively fluorescent apoptotic hepatic cells or neutrophils. The ability of these monocytes to clear apoptotic cells was then assessed through fluorescent microscopy and flow cytometry. Both techniques were used to show the increased capacity of monocytes for dead cell clearance following stimulation with secretory leukocyte protease inhibitor.

#### THE MECHANISM OF DEAD CELL CAPTURE BY PROFESSIONAL AND NON-PROFESSIONAL EFFEROCYTES

Phagocytes express several receptors to recognize and subsequently clear dying cells from the tissues (49–51). In the case of professional phagocytes (e.g., macrophages) multiple apoptotic and necrotic cell receptors have been characterized and these remain relevant in the liver (37, 52) (**Table 1**). First described in 1992, it is now widely accepted that apoptotic cells are recognized through their expression of PtdSer on the outer leaf of the plasma membrane (53, 54). Several receptors directly recognize PtdSer, many of which are expressed by professional phagocytes (55). These include stabilin-1, stabilin-2, brainspecific angiogenesis inhibitor 1 (BAI1), and RAGE, as well as the TIM family of transmembrane glycoproteins, including TIM-1, -3, and -4 (56–61). Mammary, alveolar and mesangial epithelia recognize apoptotic cells *via* the PSR, CD36, the vitronectin receptor αvβ3, and CD91 (62–64). Of note, molecules that bind PtdSer such as high-mobility group box 1 (HMGB1) can also downregulate apoptotic cell clearance (65, 66).

It is common for PtdSer to be recognized in complex with certain bridging molecules. Some of the mostwell-studied PtdSer receptors, the TAM tyrosine kinases (Tyro3, Axl, and MerTK) work in this manner (102); notably, hepatocytes express Axl but not Tyro3 or MerTK (103). The earliest known examples of these are Gas6 and Protein S (104, 105). Gas6 is universally recognized by TAM receptors, whereas Protein S, which is expressed in hepatocytes, is not recognized by Axl. Similarly, integrins αvβ3 and αvβ5 have been shown to promote efferocytosis through the recognition of PtdSerin complex with lactadherin, also known as milk fat globule EGF factor 8 (MFG-E8) (67, 68, 106).

The entirety of apoptotic cell recognition does not lie with the detection of PtdSer expression. It was shown that Tubby protein and its relative Tubby-like protein 1 (TuLP1), which do not bind PtdSer, specifically localize at the surface of apoptotic cells and could act as TAM receptor bridging molecules in a similar manner to Gas6, which in turn promoted apoptotic cell clearance (89). All TAM tyrosine kinases recognized TuLP1, whereas Tubby was exclusively recognized by MerTK on macrophages and retinal pigment cells. Mechanisms of immune surveillance and signaling have also been shown to contribute to apoptotic cell clearance. Components of the complement pathway have been shown to induce phagocytosis in macrophages and DCs by opsonizing apoptotic cells, including C1q and C3 (34, 78, 107). Furthermore, SIGN-R1, a mouse analog of human mannose receptor DC-SIGN, wasshown to bind apoptotic cells and induce theirlabelingwith C3 and subsequent clearance by marginal zone macrophages (92).

Recognition of apoptotic cells, although important, is not sufficient for macrophagesto engulf and clearthem. Downstream intracellular signaling is necessary for load-processing following capture. An important, highly-conserved signaling pathway has been described downstream from most common PtdSerreceptors, involving GTPase Rac1 and ELMO1-DOCK180 interactions(50).TAM-familymolecules,αvβ5 integrins andBAI1 act as docks for apoptotic cells, leading to intracellular signaling *via* this pathway (108, 109). Upon engagement of an apoptotic cell by these receptors, DOCK180 isrecruited by ELMO1 (110, 111). In complex, these proteins act as guanine exchange factors, allowing for Rac1 activation, which induces necessary cytoskeletal arrangements required for complete engulfment of the prey cell. Stabilin 1 and 2 have also been shown to activate this Table 1 | Efferocytosis receptors in professional phagocytes and tissue epithelia.

pathway through the adaptor protein GULP (109, 112, 113). Completion of apoptotic cell engulfment also commonly involves the activation of nuclear receptors. Loose nucleotides released from dying cells commonly act as "*eat-me*" signals, and can engage purigenic P2 receptors (P2X and P2Y), leading to an increased capacity for efferocytosis in macrophages (114, 115). It was recently shown that liver X receptor (LXR) was necessary for the capture and processing of apoptotic cells by macrophages and dendritic cells (116, 117). LXR responds to oxysterols found in engulfed apoptotic cells. Stimulation of LXR upregulated MerTK and anti-inflammatory cytokines IL-10 and TGF-β, while also leading to the downregulation of proinflammatory cytokines such as IL-1β, CCL2, and MARCO. A-Gonzalez and Hidalgo reviewed nuclear receptors and their role in macrophage efferocytosis recently (118). LXRα mediates fatty acid regulation in hepatocytes (119), but its role in hepatocyte efferocytosis remains to be determined.

Non-professional phagocytes, such as epithelial cells express multifunctional scavenger receptors, or molecules that exert alternative functions in other cell types. For example, TIM-1, also known as kidney injury molecule 1 (KIM-1), is known to possess multiple immune functions, including CD4+ T-cell and mast cell activation (59). However, TIM-1 was also upregulated in kidney epithelia following injury, allowing for a temporary efferocytic capability (99). Certain cell-exclusive receptors and modulators associated with apoptotic cell clearance have also

been described. Apoptotic cell clearance in the liver has been shown to involve asialoglycoprotein receptor (ASGPR) on hepatocytes (96). This ASGPR1 and ASGPR2 complex is critical forreceptor-mediated endocytosis of terminally desialylated glycoproteins and isrestricted to the liver.AutoantibodiestoASGPR have been found in patients and models of autoimmune hepatitis (120–122). Resolvin D1 is also important in liver protection from ischemia/reperfusion injury, by enhancing efferocytosis by M2-polarizing macrophages (123). Furthermore, phagocytosis by retinal pigment cells, although mostly conducted through MerTK, was shown to be increased through recognition of ATP-binding cassette subfamily F member 1 (ABCF1) released from apoptotic photoreceptor outer segments (124). Overall, it appears that although the broad function of recognition and engulfment of apoptotic cells is conserved throughout many cell types, multiple mechanisms exist that conduct these processes across phagocytes, both homo- and heterotypically.

#### REGULATION OF EFFEROCYTOSIS

External stimuli are pertinent for regulation of dead cell clearance by efferocytosis. As such, "*find-me*" signals released by apoptotic cells are often necessary for the guidance of efferocytes to their prey (53). The best-characterized examples of these are extracellular nucleotides (115). It was shown that upon caspase 3/7 activation in apoptotic cells, ATP and UTP released from apoptotic cells could recruit monocytes/macrophages through recognition by P2Y2. Conversely, molecules with the opposite effect known as "*don't eat-me*" signals have also been described. CD47 is the most notable, having been shown to provide resistance to clearance by macrophages on malignant cells and more recently on atherosclerotic plaques (125, 126). Similar "findme" signals may be utilized by non-motile phagocytes, which extend protrusions to collect apoptotic cells for clearance but are restricted to targets within their tissue niche.

Due to the influence of dying cells on the immune response, cytokine and growth factor stimulation of both professional and non-professional phagocytes can regulate their capacity to clear dead cells. Apoptotic T-cell lymphomas release sphingosine-1-phosphate, a bioactive lipid often involved in immune cell recruitment, leading to the recruitment of macrophages and monocytes (127). Similarly, certain chemokines, tasked with immune cell recruitment have also been shown to increase phagocyte recruitment to areas of apoptotic cells. CX3CL1 (fractalkine) was shown to recruit macrophages to its source, apoptotic Burkitt lymphoma cells (128).

Multiple cytokines have varying effects on efferocytosis (129). Most notably, secretion of IL-3 and IL-14 increased efferocytosis in macrophages through activation of PPAR and increase in CD36 expression (130, 131). IL-4 has been reported to upregulate expression of other PtdSer-receptors such as stabilin 1and 2 (61). IL-10 and TGF-β can also increase efferocytes by macrophages (132, 133). In contrast, pro-inflammatory cytokines reduce the capacity for dead cell engulfment: TNF-α has been shown to inhibit efferocytosis in macrophages (134) and both IFN-γ secretion and receptiveness were reversely correlated with antiinflammatory cytokines and receptors including IL-4 and TIM receptors (59, 129, 135). However, this was not always the case for these cytokines. Both TNF-α and IFN-γ have been shown to increase LOX-1, which may recognize apoptotic cells by LDLlabeled PtdSer. Furthermore, IFN-γ activation of macrophages, in the absence of other pro-inflammatory stimuli, was shown to increase apoptotic uptake (135).

The ability of phagocytes to clear dead cells is also subject to regulation. This is the result of alterations in gene expression, which can function as negative feedback following initial engulfment of dying cells. For example, it has been shown that macrophages, upon engulfing apoptotic cells can undergo a form of activation and reprograming (136). As well as skewing the macrophage to a more anti-inflammatory phenotype, which in turn promotes inflammatory resolution, both mouse and human macrophages can upregulate CXCR4 during efferocytosis, which in turn encourages their recruitment to draining lymph nodes (137). These macrophages were also shown to subsequently reduce their efferocytosis capacity. Thus, apoptotic cells can reduce local levels of efferocytosis as well as promote them.

More recently macrophages were shown to regulate efferocytosis in surrounding non-circulating phagocytes, such as phagocytic airway epithelial cells (138). In response to IL-4 and IL-13, which are secreted by epithelia and stimulated Th2 cells, macrophages upregulated secretion of both insulin-like growth factor 1 (IGF-1) and microvesicles containing anti-inflammatory signals. Both microvesicles and IGF-1, in turn, fed back to epithelia, causing a reduction of apoptotic cell clearance in favor of microvesicle uptake.

In the context of the liver, some of the mechanisms described for the regulation of efferocytosis apply to circulating and resident macrophages/monocytes. Further, the neuronal guidance protein netrin-1 has been shown to promote resolution of ischemia/reperfusion injury, in part by increasing the capacity of Kupffer cells to engulf apoptotic cells (139). The same molecule was shown to promote liver regeneration (139). In a mouse model of colon carcinoma metastasis in the liver, intercellular cell adhesion molecule 1-deficient macrophages cocultured with tumor cells showed increased efferocytosis dependent on phosphatidylinositol 3 kinase (140).

Environmental factors can also affect phagocytosis, and this extends to the clearance of dead cells; studies in human skin have demonstrated that ethanol can reduce phagocytic function (141), and there have been reports on increased phagocytosis in ethanol-fed rats, which was modulated by diet (142). Hepatocyte phagocytosis of apoptotic cells was decreased in ethanol-fed rats compared to controls, therefore the effects of ethanol on efferocytosis may be cell typedependent (97). It is unclear whether professional phagocytes play a role in the regulation of efferocytosis by hepatocytes and liver endothelial cells.

#### THE IMPACT OF EFFEROCYTOSIS BY TISSUE EPITHELIA

Non-professional efferocytes are important throughout all developmental stages of an organism and can take over the clearance of apoptotic cells in the absence of professional phagocytes (143). This was confirmed in PU.1 knockout mice that lack macrophages, and the removal of apoptotic cells required for foot-limb development was instead performed by mesenchymal cells (144). Non-professional phagocytes therefore contribute to efferocytosis, even at the earliest stages of development.