Efferocytosis Signaling in the Regulation of Macrophage Inflammatory Responses Michael R

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Efferocytosis Signaling in the Regulation of Macrophage Inflammatory Responses Michael R. Elliott, Kyle M. Koster and Patrick S. Murphy This information is current as J Immunol 2017; 198:1387-1394; ; of September 25, 2021. doi: 10.4049/jimmunol.1601520 http://www.jimmunol.org/content/198/4/1387 Downloaded from References This article cites 126 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/198/4/1387.full#ref-list-1

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Efferocytosis Signaling in the Regulation of Macrophage Inflammatory Responses Michael R. Elliott, Kyle M. Koster, and Patrick S. Murphy Since the pioneering work of Elie Metchnikoff and the modulate macrophage immune responses in the context of discovery of cellular immunity, the phagocytic clearance tissue inflammation. Although we primarily focus on mac- of cellular debris has been considered an integral com- rophage responses in this review, it is important to emphasize ponent of resolving inflammation and restoring func- that many different types of hematopoietic and nonhematopoietic tion of damaged and infected tissues. We now know cell types can carry out efferocytosis and that apoptotic cells that the phagocytic clearance of dying cells (efferocytosis), can exert important and sometimes cell type–specific im- particularly by macrophages and other immune phago- munoregulatory consequences on these phagocytes (reviewed cytes, has profound consequences on innate and adap- in Refs. 4–6). Downloaded from tive immune responses in inflamed tissues. These Cell death in inflamed tissues immunomodulatory effects result from an array of mo- Cell death is a universal feature of infected and damaged lecular signaling events between macrophages, dying tissues. Cell death can occur by unregulated, accidental means cells, and other tissue-resident cells. In recent years, such as mechanical or osmotic lysis, particularly in the early many of these molecular pathways have been identified stages of inflammation. However, inflammation-associated cell http://www.jimmunol.org/ and studied in the context of tissue inflammation, help- death can also result from the activation of specific biochemical ing us better understand the relationship between effer- pathways (e.g., apoptosis, necroptosis) (7). Apoptosis is the ocytosis and inflammation. We review specific types of most prominent mechanism of programmed cell death seen in efferocytosis-related signals that can impact macro- inflamed tissues for both hematopoietic and nonhematopoietic phage immune responses and discuss their relevance cells (1, 8). The stimuli that trigger apoptosis within inflamed to inflammation-related diseases. The Journal of Im- tissues vary widely depending on the type of inflammation, and munology, 2017, 198: 1387–1394. can be due to pathogen-derived, host-derived, or iatrogenic stimuli. However, it is important to note that inflammation- by guest on September 25, 2021 associated apoptosis is most often multifactorial, and in most issue injury or infection leads to the prompt activation inflammatory settings the precise cellular and molecular mecha- of a local inflammatory response to mediate pathogen nisms of apoptosis induction are poorly understood. Nonetheless, clearance and limit tissue damage. The proper ter- T it is clear that apoptotic cell death plays an integral role in the mination of this inflammatory cascade is important to allow outcome of tissue inflammation. execution of tissue repair and resolution responses required to Sepsis is one of the best-studied examples in which apoptosis restore normal tissue function. Improper resolution can result has clear and profound consequences on the resolution of in excessive scarring and organ damage as well as chronic inflammation. A life-threatening condition responsible for inflammation and loss of self-tolerance that contributes to ∼250,000 United States deaths annually, sepsis can occur disease states such as cardiovascular disease, cancer, and auto- when a localized infection fails to properly resolve, leading to immunity (1). In healthy, immune-competent hosts, most in- systemic inflammation, organ failure, and death (2, 3, 9). In flammatory episodes are self limiting, with endogenous host humans and mice, sepsis is associated with widespread apo- mechanisms successfully orchestrating the onset and resolution ptosis in lymphoid organs, causing dramatic reductions in of inflammation. The identification and molecular character- hematopoietic cell numbers, including thymic T cells, bone ization of these endogenous resolution pathways have been a marrow B cells, and peripheral lymphocytes (7, 10). This promising and intense area of research in recent years (1–3). In depletion can cause persistent lymphopenia that contributes this review, we will discuss how some of the signals that reg- to mortality (11). In mice, blockade of apoptosis using caspase ulate apoptotic cell clearance (efferocytosis) can also function to inhibitors or overexpression of antiapoptotic genes improves

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Immunology, University of Rochester School of Medicine and Dentistry, 601 Biology and Immunology, University of Rochester School of Medicine and Dentistry, Elmwood Avenue, Box 609, Rochester, NY 14642. E-mail address: michael_elliott@urmc. Rochester, NY 14642 rochester.edu Received for publication August 31, 2016. Accepted for publication September 23, Abbreviations used in this article: DC, dendritic cell; LAP, LC3-associated phagocytosis; 2016. lysoPC, lysophosphatidylcholine; NR, nuclear receptor; PtdSer, phosphatidylserine; S1P, sphingosine-1-phosphate; WT, wild-type. This work was supported by National Institutes of Health Grant R01 AI114554 (to M.R.E.) and in part by National Institutes of Health Grants T32 GM007356 Ó (to K.M.K.) and T32 GM007285 (to P.S.M.). Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 Address correspondence and reprint requests to Dr. Michael R. Elliott, Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601520 1388 BRIEF REVIEWS: EFFEROCYTOSIS SIGNALING IN MACROPHAGE INFLAMMATION survival following polymicrobial sepsis (12, 13). Similarly, of uncleared apoptotic cells in situ along with spontaneous most myeloid cell populations are also depressed in sepsis inflammation and autoimmune disease (reviewed in Refs. 6, (e.g., monocytes, macrophages, dendritic cells [DC]), al- 26, 27). However, efferocytosis can also directly alter the in- though neutrophil survival is increased (14). Consequently, flammatory signaling pathways in the engulfing macrophage. sepsis-associated leukocyte apoptosis is thought to tran- In this review, we will discuss how many of the signals that siently impair immunity and contribute to the generation of mediate the three stages of efferocytosis also play important an immunosuppressed state known as compensatory anti- roles in driving the immunomodulatory effects of apoptotic inflammatory response syndrome (15). The extent to which cells on macrophages. immunomodulatory effects of efferocytosis contribute to sus- tained immune suppression is an important but poorly un- Find-me signaling in inflammation derstood area of sepsis research (16). For phagocytosis to occur, phagocytes must first find a cell Leukocyte apoptosis is also a characteristic feature of self- undergoing apoptosis. This find-me stage of efferocytosis is limiting inflammation resulting from acute, localized, and mediated by the release of numerous soluble factors that attract moderate injury or infection. However, unlike sepsis, apoptotic macrophages to the site of cell death (6, 28, 29). Key find-me cells are thought to be beneficial in self-resolving inflammation signals identified to date include triphosphate nucleotides by helping to reprogram tissue macrophages from a proin- (ATP, UTP) (30, 31), the chemokine CX3CL1 (32), and the flammatory to a proresolution state (17). Tissue-resident signaling lipids lysophosphatidylcholine (lysoPC) and sphingosine- macrophages are key immune sentinels and thus are among 1-phosphate (S1P) (33–35). Although all of these factors can Downloaded from the first immune cells to respond to tissue damage by pro- stimulate macrophage migration to apoptotic cells, the rele- ducing cytokines that initiate and orchestrate the recruitment vance of individual find-me signals in efferocytosis depends of granulocytes from the blood into the tissue (18). Neutro- on many factors, including phagocyte and apoptotic cell type phils typically accumulate in significant numbers within as well as the apoptotic stimuli and the stage of apoptosis minutes to a few hours depending on the nature and severity being studied (reviewed in Ref. 36). As discussed below, some of the injury. Once in the tissue, most neutrophils rapidly of these find-me signals also function as key modulators of http://www.jimmunol.org/ undergo apoptosis and begin to accumulate as uncleared ap- macrophage inflammatory responses. optotic cells. The time at which viable neutrophil numbers are Apoptotic cells release adenine and uridine nucleotides highest in the tissue is often considered the peak of a self- through caspase-3/7–activated hexameric pannexin-1 channels limiting inflammatory episode (17). Importantly, the maxi- on their surface (37). Extracellular nucleotides ATP and UTP mum number of uncleared apoptotic neutrophils in the tissue act as find-me signals to promote cell clearance by stimulating typically coincides with this peak, followed by macrophage- recruitment of motile phagocytes expressing P2Y purinergic mediated efferocytosis and clearance of the neutrophils during receptors and also by causing upregulation of phagocytic re- the resolution stage (19). Such observations support the the- ceptors (30, 38–40). Extracellular nucleotides can also exert by guest on September 25, 2021 ory that efferocytosis of apoptotic neutrophils by resident immunomodulatory effects on macrophages via the break- macrophages is an important immune trigger for the onset of down of ATP into adenosine—a well-known and potent resolution (20, 21). However, confirmation of this theory has modulator of macrophage inflammation (41, 42). Recent been hampered by the difficulty in tracking the fate of engulfed reports have shown that during efferocytosis, Gs-linked A2a endogenous apoptotic cells. Nonetheless, there is abundant and A2b adenosine receptors on macrophages mediate sup- evidence that apoptotic cells can suppress macrophage in- pression of proinflammatory cytokines(e.g.,CXCL1,CXCL2) flammatory responses in vitro and that failure to clear apoptotic and upregulation of proresolution factors (e.g., Nr4a1, Thbs1) cells exacerbates inflammation in vivo, indicating efferocytosis (43, 44) (Fig. 1). Interestingly, the precise mechanisms respon- plays a crucial role in modulating the inflammatory response sible for generating extracellular adenosine during efferocytosis of macrophages (and other phagocytes) to promote resolu- are poorly understood. Although adenosine can be released tion(5,6,22). directly from macrophages (43, 45), in many tissue settings the accumulation of extracellular adenosine is largely due to Homeostatic cell clearance the hydrolysis of extracellular adenine nucleotides (i.e., ATP, Approximately 1 3 1010 cells undergo apoptosis daily in the ADP, AMP) by ecto-enzymes such as CD39 (ATP/ADP→AMP) human body; however, few free apoptotic cells are seen at and CD73 (AMP→adenosine) (41, 42). How these ecto- homeostasis, indicating the presence of highly efficient enzymes contribute to adenosine generation and immune efferocytosis mechanisms (23). This efficiency depends on the modulation of macrophages during efferocytosis remains an execution of the three main efferocytosis signaling programs open question. depicted in Fig. 1: 1) find-me signaling—chemoattractant- The chemokine CX3CL1 was identified as an apoptotic mediated recruitment of phagocytes to apoptotic cells, 2) eat- find-me signal by Truman and colleagues (32), and since has me signaling—receptor-mediated recognition and engulfment been implicated in cell clearance and tissue repair in a number of apoptotic cells, and 3) postengulfment signaling—signals of tissue settings. Apoptotic cell–derived CX3CL1 stimulates related to the phagolysosomal processing of engulfed cellular ma- recruitment of CX3CR1+ phagocytes to clear apoptotic debris terial. The most obvious anti-inflammatory effect of efferocytosis is in lymphoid tissues and the brain (32, 46). Additionally, the physical sequestration of dying cells to limit the release of CX3CL1 from apoptotic cells enhances efferocytosis by stimu- intracellular damage-associated molecular patterns that can lating phagocytes to express the eat-me ligand MFG-E8 (47–49). drive inflammation (7, 24, 25). Indeed, there is now very In the context of tissue repair, CX3CL1–CX3CR1 signaling has strong evidence that disrupting homeostatic efferocytosis in been shown to play a beneficial role by reducing expression of mice, either genetically or chemically, can lead to accumulation inflammatory cytokines and enhancing expression of prosurvival The Journal of Immunology 1389 Downloaded from http://www.jimmunol.org/

FIGURE 1. Immunomodulatory roles of efferocytosis signals. Depiction of the three key stages of efferocytosis (dashed boxes) with detailed illustrations showing some of the known signaling molecules/pathways relevant to immune modulation for each of the three stages. The lower table indicates some of the key effects of these efferocytosis signaling mechanisms on the following: immune signaling in phagocytes, production of immune mediators, and some of the prominent immune outcomes of the indicated molecules/pathways. Arrows in the table indicate whether signaling via these pathways generally increases or decreases the effects listed. Please note this diagram represents only a portion of the efferocytosis signals and their immunoregulatory effects that have been described. More comprehensive lists of these signaling pathways can be found in Refs. (6, 61). MMP, matrix metalloprotease; PS, phosphatidylserine (PtdSer); RTK, receptor tyrosine kinase. by guest on September 25, 2021

(e.g., BCL-2) and antioxidant factors (HO-1) (49–51). Thus, Eat-me signaling in inflammation release of CX3CL1 from dying cells not only serves to promote Macrophages primarily recognize apoptotic cells via high levels cleanup of damaged tissues, but also aids in driving expression of of surface phosphatidylserine (PtdSer). Normally confined to genes that are beneficial to the restoration of tissue homeostasis. the inner leaflet of viable cells, PtdSer is enriched on the Lipid find-me signals also double as chemoattractants and im- exofacial side of the membrane as a result of caspase-mediated mune regulators during efferocytosis. S1P is released by apoptotic changes in the activity of several key phospholipid transport cells through the caspase-dependent upregulation of S1P kinases 1 enzymes (reviewed in Ref. 59). At least 12 PtdSer efferocytosis and 2 (34, 35). Chemotaxis of myeloid cells to S1P is mediated by receptors have been identified to date and comprise a struc- the S1P family of G-protein–coupled receptors (S1PR1–5) (52). turally heterologous group of surface proteins that bind either Beyonditsroleasamacrophage chemoattractant, Brune¨ and directly to PtdSer or indirectly via recognition of soluble colleagues (53–55) found that S1P derived from apoptotic cells PtdSer-binding opsonins (reviewed in Refs. 6, 60). In 2002, provokes an antiapoptotic and anti-inflammatory gene ex- Henson and colleagues (61) first showed that PtdSer on ap- pression program in macrophages, characterized by suppression optotic cells was not only a critical eat-me ligand, but also that of TNF and IL-12 along with increased production of IL-10, PtdSer recognition by macrophages has profound immuno- vascular endothelial growth factor, and PGE2.Moreover,S1PR modulatory effects on macrophages and other phagocytes. signaling in macrophages promotes an alternative M2-like anti- Since this time, many efferocytic receptors have been identi- inflammatory phenotype, including increased cAMP and fied and subsequently found to play a role in regulating cyclooxygenase-2 and suppression of NF-kBsignaling(52).More macrophage immune responses (Fig. 1). recently, Luo et al. (56) reported that apoptotic cell–derived S1P The four best-studied bona fide PtdSer efferocytosis receptors can stimulate erythropoietin autocrine signaling in macro- are as follows: BAI, TIM, Stabilin, and CD300. The G-protein– phages that stimulates activation of the M2-promoting nuclear coupled receptor BAI1 directly binds PtdSer via a series of receptor PPAR-g. Like S1P, lysoPC is also a potent chemo- N-terminal thrombospondin repeats and subsequently stimu- attractant find-me signal for monocytes and macrophages lates actin polymerization and phagocytosis via recruitment and during efferocytosis (33), and deletion of a putative lysoPC activation of the Elmo-Dock bipartite Rac-GEF (62). Although receptor G2A in mice leads to increased tissue inflammation Bai1 expression is restricted to specific phagocyte populations and hallmarks of systemic autoimmunity (57, 58). However, it (62), it appears particularly important for efferocytosis by is presently unclear what role apoptotic cell–derived lysoPC gastric and intestinal phagocytes (63). Using a mouse model of plays in the immunoregulatory functions of G2A in vivo. colitis, Lee et al. (64) recently reported that Bai1-deficient mice 1390 BRIEF REVIEWS: EFFEROCYTOSIS SIGNALING IN MACROPHAGE INFLAMMATION displayed increased colonic inflammation, tissue damage, and inhibitory outcomes depending on their association with dif- mortality. It was also shown that Bai1 deletion in macrophages ferent cytoplasmic signaling modules. For example, although and colonic epithelial cells leads to higher levels of IL-1a,IL-6, signaling via the ITIM domain of CD300A inhibits phagocy- and TNF in the presence of apoptotic cells in vitro (64). These tosis of apoptotic cells, this domain is important for suppression findings are in line with previous studies showing that Elmo1- of inflammatory cytokines during efferocytosis (85, 86). Dock signaling not only regulates actin dynamics, but can also CD300F can both stimulate and inhibit efferocytosis depending influence the expression of inflammatory genes (65–67). on the type of phagocyte, and CD300F-deficient mice develop Presently, it is not clear how Bai/Elmo/Dock signaling alters symptoms of autoimmunity (87). CD300B signaling via the inflammatory gene expression (64). Interestingly, separate adaptor protein DAP12 positively regulates efferocytosis, and studies have shown that Elmo can localize to the nucleus (68) Voss et al. (88) recently showed that LPS treatment of macro- and interact with transcriptional machinery (66), but whether phages causes CD300B to interact with TLR4 and that this these functions are relevant to changes in macrophage gene interaction augments TLR4-dependent production of proin- expression during efferocytosis is not known. flammatory cytokines. Along these lines, the CD300F receptor The TIM family of receptors (hTIM-1, 3, 4) are type I cell was recently shown to interact directly with the common chain surface glycoproteins that bind PtdSer via N-terminal IgV of IL-4R/IL-13R to enhance production of IL-4 and thus could domains to mediate apoptotic cell engulfment (69–71). TIM-1 potentially promote M2 polarization in macrophages (89). Al- and TIM-3 possess one or more tyrosine phosphorylation sites though CD300 molecules have been shown to regulate immune in their C-terminal cytoplasmic domains that control ligand- responses in many different tissue settings, it remains to be seen Downloaded from dependent recruitment and suppression of Src family kinases exactly how CD300 signaling during efferocytosis impacts im- (70). TIM expression in murine immune phagocytes is highly mune signaling and cytokine production in macrophages during subset and tissue restricted, and mice lacking individual TIM theresolutionoftissueinflammation. molecules, although viable, display a number of organ-specific Stabilin receptors (Stab1, Stab2) are type I surface receptors immune defects (70, 72–75). In a renal ischemia/reperfusion that trigger efferocytosis through direct interaction with injury model, TIM-1 binding of PtdSer is required for apo- PtdSer (90–92). In mice, Stabilin receptors are expressed in a http://www.jimmunol.org/ ptotic cells to suppress NF-kB activation and production of number of macrophage populations (93), whereas in humans TNF, IL-6, and CCL5 in proximal tubular cells (76, 77). In Stab1 was found to be highly expressed in endothelial cells these studies, tyrosine phosphorylation of the TIM-1 cyto- and steroid-treated tissue macrophages (94). In vitro, expres- plasmic tail caused increased recruitment of the p85 subunit of sion of Stab2 in cultured cells stimulates the binding and Rac- PI3K and inhibition of NF-kB activation downstream of dependent internalization of apoptotic cells and also stimulates TLR4 (77). TIM-3 is expressed on multiple macrophage and TGF-b production (91). Interestingly, Stab1/Stab2 double- DC populations, and can evoke both immune-activating and knockout mice have shorter lifespans and increased tissue in-

immune-suppressing outcomes during efferocytosis (78). In flammation compared with wild-type (WT) mice, although by guest on September 25, 2021 TLR-stimulated CD8+ DC, TIM-3 can promote cross- these results most likely stem from the loss of hyaluronic acid presentation of corpse Ags (79). However, signaling via TIM-3 scavenging mediated by these receptors (95, 96). How Stabilin can also inhibit NF-kB and inflammatory cytokine production signaling regulates cytokine expression during efferocytosis is via tyrosine phosphorylation of theTIM-3cytoplasmictail(78). still unclear, although STAB1-mediated engulfment requires TIM-4, by contrast, has a short cytoplasmic tail that is dispensable the intracellular adaptor protein GULP, which has been shown for apoptotic cell engulfment, suggesting that TIM-4 functions as to regulate cytokine production downstream of other receptors, a high-affinity PtdSer binding/tethering receptor rather than bona including LRP1 (97, 98). fide engulfment signaling receptor (80, 81). Still, Martinez et al. A number of efferocytic receptors indirectly recognize (82) recently showed that TIM-4–mediated corpse engulfment PtdSer on apoptotic cells via interaction with PtdSer-binding can trigger recruitment of specific autophagy components to the bridging proteins such as MFG-E8, CCN1, GAS6, and phagosome in a distinct form noncanonical autophagy termed protein S (ProS1) (6, 60). Upon binding to PtdSer on apo- LC3-associated phagocytosis (LAP). Subsequently, Green and ptotic cells, MFG-E8 and CCN1 are recognized by avb3/5 colleagues (82) reported that genetic disruption of LAP in mye- integrin/vitronectin receptors on phagocytes, leading to Rac loid cells (via deletion of NOX2 or RUBCN) leads to a chronic activation and corpse internalization (99, 100). It is clear that autoinflammatory disorder and development of systemic lupus av-mediated engulfment of MFG-E8–opsonized apoptotic erythematosus–like disease in mice. In these studies, LAP-deficient cells is important for limiting tissue inflammation and macrophages stimulated with apoptotic cells in vitro were found maintenance of self-tolerance in mice (99, 101). However, it to produce much higher levels of inflammatory cytokines (IL-1b, is less clear whether avb3/5 signaling directly regulates cyto- IL-6, CXCL10) and lower levels of IL-10 compared with LAP- kine production in macrophages during efferocytosis. Studies sufficient macrophages (83). Given that TIM-4–deficient mice using mimetic RGD peptides (Arg-Gly-Asp) to manipulate also develop hallmarks of tissue inflammation and autoimmunity av-mediated apoptotic cell recognition have reported signifi- (73, 74), it will be important to delineate the precise roles of cant effects on macrophage TNF and IL-10 production (102, TIM-4 in conventional efferocytosis versus LAP in shaping 103). By contrast, studies using avb3/5 blocking Abs or macrophage inflammatory responses to apoptotic cells. macrophages deficient in b3 or b5 did not find substantial Of the seven human CD300 genes, three have been shown effects on cytokine production during efferocytosis (104, to bind PtdSer and modulate efferocytosis, as follows: CD300A, 105). Still, it is important to note that the role of av integrins CD300B, and CD300F (84). However, the immunomodula- in efferocytosis is quite complex owing to its interactions with tory effects of CD300 receptors during efferocytosis are not multiple b-chains (i.e., b1, b6, b8) and the formation of straightforward, as these receptors can trigger either activating or signaling complexes with other efferocytosis molecules, such The Journal of Immunology 1391 as CD36, thrombospondin, and HMGB1 (106, 107). Thus, finding from these studies is that NR activation, both homeo- av integrins play key roles in macrophage adhesion, motility, statically and during efferocytosis, enhances transcription of and phagocytosis, but more investigation is required to define engulfment-related genes in macrophages, including PtdSer re- their contribution to immune signaling during efferocytosis. ceptors, PtdSer-binding soluble proteins, and key intracellular The Tyro3, Axl, Mer (TAM) tyrosine kinases are another engulfment-signaling molecules (Fig. 1). Accordingly, mice family of efferocytosis receptors that recognize PtdSer on lacking one or more of these NRs display defective clearance of apoptotic cells indirectly through binding to the PtdSer- apoptotic cells, enhanced tissue inflammation, and autoimmu- binding serum proteins GAS6 and ProS1. All three TAM nity (120–122). However, it is important to note that although receptors are capable of mediating efferocytosis, with Axl and internalization of apoptotic cells can modulate the activity of Mer being particularly important for efferocytosis by DC and multiple NRs, we presently do not know the cellular source or macrophages (108, 109). Disruption of TAM signaling in precise molecular nature of the NR-modulating ligands relevant vivo leads to increased levels of uncleared apoptotic cells in to efferocytosis. multiple tissues, and TAM-deficient mice display exacerbated In addition to promoting phagocytosis, NRs are involved in the responses to apoptotic cells and inflammatory stimuli in vivo regulation of macrophage inflammation during efferocytosis (108, 110, 111). At the molecular level, recognition of Gas6/ (119). For example, treatment of LXR-a/b–deficient macro- ProS1-opsonized apoptotic cells causes TAM receptor di- phages with apoptotic cells does not induce TGF-b and IL-10 merization leading to Rac activation and apoptotic cell en- production nor suppress IL-1b and IL-12 production like their gulfment (112). In addition, TAMs have been shown to WT counterparts (121). Similarly, macrophages lacking PPAR-d Downloaded from function as key negative regulators of inflammation and fail to produce IL-10 and produce more TNF and IL-12 than adaptive immunity by controlling multiple immune signaling WT macrophages when cultured with apoptotic cells (120). pathways and gene expression programs. In the context of Also, macrophages from mice lacking PPAR-g andretinoidX efferocytosis, signaling via Axl and Mer can directly suppress receptor-a are refractory to the immunosuppressive effects of TLR- and type I IFN-driven inflammatory signaling pathways apoptotic cells in vitro (122). More recently, the nuclear receptor via distinct mechanisms. In DC, activation of Mer by apo- Nr4a1 was found to be rapidly upregulated in macrophages http://www.jimmunol.org/ ptotic cells inhibits IKK activity downstream of TLR4, lead- cultured with apoptotic cells, and Nr4a1-deficient macrophages ing to reduced NF-kB binding at the Tnf promoter (113). In cultured with apoptotic cells showed enhanced NF-kBactiva- a separate study, Sharif et al. (110) reported that suppression tion and IL-12 production compared with WT macrophages of TLR-driven TNF by apoptotic cells required Axl-mediated (44, 123). Interestingly, Nr4a1 upregulation by apoptotic cells upregulation of the Twist transcriptional repressors that bind can occur via adenosine activation of the A2a receptor, sug- E-box elements and inhibit Tnf promoter activity. Interest- gesting regulation of this NR could be stimulated by nucleotide ingly, TAM receptors also function as negative regulators of find-me signals as well as via phagosomal signaling (44, 124).

type I IFN-induced inflammatory signaling in DC by sup- by guest on September 25, 2021 pressing IFN-a–mediated STAT1 signaling and by upregulation Conclusions of the E3 ubiquitin ligases suppressor of cytokine signaling 1 and The discovery of the immunomodulatory activity of apoptotic 3 (111). Presently, the precise mechanisms regulating these re- cells on macrophages nearly 20 y ago transformed our view of sponses and what roles they play in efferocytosis-mediated im- efferocytosis from a simple garbage disposal process to one mune modulation remain to be fully defined. Nevertheless, it is capable of actively shaping immune responses and influencing clear that in macrophages and DC TAMs are key mediators of tissue restorative programs. This paradigm shift has led to both apoptotic cell phagocytosis and anti-inflammatory signal- promising new therapeutic avenues built on manipulating ing. As such, TAMs are very attractive targets for modulation of efferocytosis-related processes, as evidenced by the ongoing efferocytosis responses in controlling innate and adaptive im- trials of anti-PtdSer Abs in several clinical settings (125, 126). mune responses. The efferocytosis mechanisms highlighted in this work illus- trate the ability of macrophages to engage specific molecular Postengulfment signaling in inflammation pathways that control both phagocytosis and immune sig- The engulfment of apoptotic cell by macrophages results in the naling. This linkage supports the idea that the process of dead acquisition of substantial quantities of excess cellular materials cell clearance is not simply an end unto itself, but rather that such as lipids, carbohydrates, proteins, and nucleic acids, and the efferocytosis process provides key physiologic status in- macrophages can adjust to this increased metabolic load by formation regarding cell death and tissue health to the im- activating degradation and efflux pathways (114). In addition, mune system via macrophages (and other phagocytes). a number of recent studies have shown that engulfment of Finally, it is important to note that although efferocytosis apoptotic cells engages multiple metabolic sensing pathways signaling is often framed as a chronological three-step process in macrophages that play important roles in controlling (Fig. 1), physiologic efferocytosis by macrophages almost phagocytosis and immune signaling (115–118). Among these certainly involves the simultaneous integration of many dif- metabolite-sensing mechanisms, the nuclear receptor (NR) ferent efferocytosis signaling processes. As such, understand- family of transcriptional regulators stands as the best-studied ing how the spatiotemporal dynamics and potential synergistic link between sensing of ingested apoptotic cells and the mac- relationships of these different signaling pathways affect cell rophage efferocytosis machinery. Studies using genetic and clearance and immunity stands as an important, albeit very pharmacologic manipulation have identified multiple NR family challenging, area for future studies. Considering the ubiquity of members as critical regulators of macrophage efferocytosis in cell death during inflammation and the inherent signaling vitro and in vivo, including LXRa, LXRb, PPARg, PPARd,and complexities of inflamed tissues, major mechanistic advances in RXRa (reviewed in Refs. 6, 119). 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