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Skeletal muscle repair is initiated by local inflammation and involves the engulfment of dead cells (efferocytosis) by infiltrating macrophages at the injury site. Macrophages orchestrate the whole repair program, and efferocytosis is a key event not only for cell clearance but also for triggering the timed polarization of the inflammatory phenotype of macrophages into the healing one. While pro-inflammatory cytokines produced by the inflammatory macrophages induce satellite cell proliferation and differentiation into myoblasts, healing macrophages initiate the resolution of inflammation, angiogenesis, and extracellular matrix formation and drive myoblast fusion and myotube growth. Therefore, improper efferocytosis results in impaired muscle repair. Retinol saturase (RetSat) initiates the formation of various dihydroretinoids, a group of vitamin A derivatives that regulate transcription by activating retinoid receptors. Previous studies from our laboratory have shown that RetSat-null macrophages produce less milk fat globule-epidermal growth factor-factor-8 (MFG-E8), lack neuropeptide Y expression, and are characterized by impaired efferocytosis. Here, we investigated skeletal muscle repair in the tibialis anterior muscle of RetSat-null mice following cardiotoxin injury. Our data presented here demonstrate that, unexpectedly, several cell types participating in skeletal muscle regeneration compensate for the impaired macrophage functions, resulting in normal muscle repair in the RetSat-null mice.

Neonatal brain hypoxia-ischemia (HI) is a leading cause of morbidity and long-term disabilities in children. While we have made significant progress in describing HI mechanisms, the limited therapies currently offered for HI treatment in the clinical setting stress the importance of discovering new targetable pathways. Efferocytosis is an immunoregulatory and homeostatic process of clearance of apoptotic cells (AC) and cellular debris, best described in the brain during neurodevelopment. The therapeutic potential of stimulating defective efferocytosis has been recognized in neurodegenerative diseases. In this review, we will explore the involvement of efferocytosis after a stroke and HI as a promising target for new HI therapies.

Following myocardial infarction (MI), blood neutrophils quickly and extensively infiltrate the heart, where they are temporally polarized into pro-inflammatory (N1) and anti-inflammatory (N2) subpopulations. Neutrophil transmigration is rapidly followed by the accrual of macrophages (MACs), which are believed to undergo local phenotypic transformations from pro-inflammatory to pro-healing MACs that mediate inflammation resolution. We hypothesized that N2 neutrophils can reprogram MACs toward a healing phenotype with increased efferocytosis capacity. To examine this, human neutrophils isolated from healthy subjects were polarized in N1 and N2 neutrophils, and their secretome was added to human MACs derived from THP monocytes. The impact of neutrophil factors on macrophages was investigated using qPCR, ELISA, Western blot, immunofluorescence, or an efferocytosis assay. The results show that the MACs exposed to N2 neutrophil secretome exhibited (i) increased expression of the anti-inflammatory molecules CD206, TGF-β, and IL-10 and the nuclear factors associated with reparatory macrophages (PPARγ, Nur77, and KLF4); (ii) enhanced expression of efferocytosis receptors (MerTK, CD36, CX3CR1, and integrins αv/β5) and of the bridge molecules Mfage8 and Gas6; and (iii) enhanced efferocytosis. In conclusion, factors released by N2 neutrophils induce a pro-healing phenotype of MACs by upregulating anti-inflammatory molecules and efferocytosis receptors and ensuing the efferocytosis capacity. The data suggest that molecular therapy to foster N2 polarization, which boosts macrophages’ pro-healing phenotype, could be a promising strategy to speed up inflammation resolution and tissue repair.

Apoptosis and subsequent removal of dead cells are an essential part of wound healing. Macrophages phagocytize apoptotic cells (efferocytosis) and contribute to the resolution of inflammation. However, their participation in fibrogenesis and the mechanisms of influence on this process remain unclear. In the present study, we focused on the fibrogenic properties of human monocyte-derived macrophages polarized in the M2 direction by interaction with apoptotic cells. We studied their influence on the proliferation ([3H]-thymidine incorporation), differentiation (by the expression of α-SMA, a myofibroblast marker) and collagen-producing activity (ELISA) of dermal fibroblasts compared to classically (LPS) and alternatively (IL-4) activated macrophages. Macrophages polarized by the interaction with apoptotic cells had a unique phenotype and profile of produced factors and differed from the compared macrophage subtypes. Their conditioned media promoted the proliferation of dermal fibroblasts and the expression of α-SMA in them at the level of macrophages stimulated by IL-4, while the stimulating effect on the collagen-producing activity was more pronounced compared to that of the other macrophage subtypes. Moreover, they are characterized by the high level of production of pro-fibrotic factors such as TIMP-1, TGF-β1 and angiogenin. Taken together, M2-like macrophages polarized by efferocytosis demonstrate in vitro pro-fibrotic activity by promoting the functional activity of dermal fibroblasts and producing pro-fibrotic and pro-angiogenic factors.

Neutrophils are highly abundant circulating leukocytes that are amongst the first cells to be recruited to sites of infection or sterile injury. Their ability to generate and release powerful cytotoxic products ties with their role in host defence from bacterial and fungal infections. Neutrophilic inflammation is tightly regulated to limit the amount of ‘bystander injury’ caused. Neutrophils were in the past regarded as short-lived, indiscriminate killers of invading microorganisms. However, this view has changed quite dramatically in recent years. Amongst other insights, neutrophils are now recognised to also have important anti-inflammatory functions that are critical for the resolution of inflammation and return to homeostasis. This minireview focusses on anti-inflammatory neutrophil functions, placing a particular focus on recent findings linked to neutrophil cell death, several types of which may be anti-inflammatory (apoptosis, secondary necrosis, and neutrophil extracellular traps). These are discussed together with features that may further promote the clearance of dead cells by efferocytosis and reprogramming of macrophages to promote resolution and repair.

The clearance of apoptotic cancer cells by macrophages, known as efferocytosis, fuels the bone-metastatic growth of prostate cancer cells via pro-inflammatory and immunosuppressive processes. However, the exact molecular mechanisms remain unclear. In this study, single-cell transcriptomics of bone marrow (BM) macrophages undergoing efferocytosis of apoptotic prostate cancer cells revealed a significant enrichment in their cellular response to hypoxia. Here, we show that BM macrophage efferocytosis increased hypoxia inducible factor-1alpha (HIF-1α) and STAT3 phosphorylation (p-STAT3 at Tyr705) under normoxic conditions, while inhibitors of p-STAT3 reduced HIF-1α. Efferocytosis promoted HIF-1α stabilization, reduced its ubiquitination, and induced HIF-1α and p-STAT3 nuclear translocation. HIF-1α stabilization in efferocytic BM macrophages resulted in enhanced expression of pro-inflammatory cytokine MIF, whereas BM macrophages with inactive HIF-1α reduced MIF expression upon efferocytosis. Stabilization of HIF-1α using the HIF-prolyl-hydroxylase inhibitor, Roxadustat, enhanced MIF expression in BM macrophages. Furthermore, BM macrophages treated with recombinant MIF protein activated NF-κB (p65) signaling and increased the expression of pro-inflammatory cytokines. Altogether, these findings suggest that the clearance of apoptotic cancer cells by BM macrophages triggers p-STAT3/HIF-1α/MIF signaling to promote further inflammation in the bone tumor microenvironment where a significant number of apoptotic cancer cells are present.

Every day, billions of our cells die and get cleared without inducing inflammation. When, clearance is improper, uncleared cells undergo secondary necrosis and trigger inflammation. In addition, proper efferocytosis would be required for inducing resolution of inflammation, thus clearance deficiencies in the long term lead to development of various chronic inflammatory diseases. Increasing evidence indicates that obesity, itself being a low-grade inflammatory disease, predisposes to a variety of other chronic inflammatory diseases. Previous studies indicated that this later might be partially related to an impaired efferocytosis induced by increased uptake of circulating saturated fatty acids by macrophages in obese people. Here, we show that palmitate inhibits efferocytosis by bone marrow-derived macrophages in a dose-dependent manner. Palmitate triggers autophagy but also activates an energy-sensing mTORC1/ROCK1 signaling pathway, which interferes with the autophagosome–lysosome fusion, resulting in accumulation of the cellular membranes in autophagosomes. We propose that lack of sufficient plasma membrane supply attenuates efferocytosis of palmitate-exposed macrophages. AMP-activated protein kinase activators lead to mTORC1 inhibition and, consequently, released the palmitate-induced efferocytosis block in macrophages. Thus, they might be useful in the treatment of obesity not only by affecting metabolism thought so far. ROCK1 inhibitors could also be considered.

Clearance of apoptotic cells by bone marrow-derived macrophages differentiated from monocytes plays a central role in the resolution of inflammation, as the conversion of pro-inflammatory M1 macrophages to M2 macrophages that mediate the resolution process occurs during efferocytosis. Thus, proper efferocytosis is a prerequisite for proper resolution of inflammation, and failure in efferocytosis is associated with the development of chronic inflammatory diseases. Previous studies from our laboratory have shown that (13R)-all-trans-13,14-dihydroretinol (DHR), the product of retinol saturase, acting from day 4 of monocyte differentiation enhances the efferocytosis capacity of the resulted macrophages. Loss of retinol saturase in mice leads to impaired efferocytosis, and to development of autoimmunity. In the present paper, we report that in differentiating monocytes DHR, retinol, and all-trans retinoic acid all act directly on retinoic acid receptors and enhance the clearance of apoptotic cells by upregulating the expression of several efferocytosis-related genes. The effect of retinoids seems to be mediated by bone morphogenetic protein (BMP)-2, and the Smad3 transcription factor. In addition, retinoids also upregulate the expression of the vitamin D receptor and that of vascular endothelial growth factor A, indicating that altogether retinoids promote the generation of a pro-reparative M2 macrophage population during monocyte differentiation.

Mesenchymal stromal cells (MSCs) are largely known for their immune-suppressive capacity, hence, their common use in the control of unwanted inflammation. However, novel concepts related to their biology, combined with the urgent need to identify MSC subpopulations with enhanced suppressive properties, drive the search for isolation protocols optimized for clinical applications. We show, in this study, that MSCs expressing high CD146 levels exhibit altered surface expression profiles of CD44 and secrete elevated levels of interleukin (IL)-6, amongst other factors. In addition, CD146hi MSCs surpass the polyclonal parental populations in inhibiting alloreactive T cells in vitro, in both a soluble- and cell-contact-dependent manner. Despite the lack of CD146hi MSC-mediated activation of peritoneal macrophages to release the suppressive factor IL-10 in vitro, their administration in animals with graft-versus-host disease alleviates inflammation and leads to 40% survival rate up to 7 weeks post-transplantation. This pronounced inhibitory property is driven by CD146-mediated in situ efferocytosis by myeloid cells. Altogether, this study provides the impetus to adopt an isolation protocol for MSCs based on a CD146 expression profile before their therapeutic use and suggests a major role played by CD146 as a novel “eat-me” signal, capable of enhancing MSC uptake by competent phagocytes.

Skeletal muscle repair is initiated by local inflammation and involves the engulfment of dead cells (efferocytosis) by infiltrating macrophages at the injury site. Macrophages orchestrate the whole repair program, and efferocytosis is a key event not only for cell clearance but also for triggering the timed polarization of the inflammatory phenotype of macrophages into the healing one. While pro-inflammatory cytokines produced by the inflammatory macrophages induce satellite cell proliferation and differentiation into myoblasts, healing macrophages initiate the resolution of inflammation, angiogenesis, and extracellular matrix formation and drive myoblast fusion and myotube growth. Therefore, improper efferocytosis results in impaired muscle repair. Retinol saturase (RetSat) initiates the formation of various dihydroretinoids, a group of vitamin A derivatives that regulate transcription by activating retinoid receptors. Previous studies from our laboratory have shown that RetSat-null macrophages produce less milk fat globule-epidermal growth factor-factor-8 (MFG-E8), lack neuropeptide Y expression, and are characterized by impaired efferocytosis. Here, we investigated skeletal muscle repair in the tibialis anterior muscle of RetSat-null mice following cardiotoxin injury. Our data presented here demonstrate that, unexpectedly, several cell types participating in skeletal muscle regeneration compensate for the impaired macrophage functions, resulting in normal muscle repair in the RetSat-null mice.

Impairment of efferocytosis in apoptotic macrophages is a known determinant of the severity of atherosclerosis and the vulnerability of plaques to rupture. The precise mechanisms involved in impaired efferocytosis are unclear. Given the well-recognized role of the inflammatory cytokine cyclophilin A (Cyp A) in modulating several atherogenic mechanisms in high-glucose primed monocytes, we investigated the role of Cyp A in macrophage efferocytosis. The efficiency of efferocytosis in RAW 264.7 macrophages grown in vitro and primed with cyclophilin A was assessed using flow cytometry and confocal assays. Cholesterol content in cells was measured using cell-based cholesterol efflux assay. Proteomic analysis and bioinformatics tools were employed to decipher the link between cyclophilin A and the known ligand receptors involved in efferocytosis. Cyclophilin A was found to impair efferocytosis in apoptotic macrophages by reducing ABCA1-mediated cholesterol efflux in foam cells derived from macrophages. Cyclophilin A-primed macrophages showed an increase in expression of the don’t-eat-me signal CD 47 and a decrease in the expression of the eat-me signal, calreticulin. Phagocytosis was restored upon silencing of cyclophilin A. New Zealand white rabbits were fed a high-fat diet, and lesions in their aortae were analyzed histologically for evidence of atherosclerosis and the expression of Cyp A, CD 47 and calreticulin, the ligand receptor involved in efferocytosis. Gene and protein expressions in aortae and macrophages were analyzed by real-time PCR and Western blotting. Cyclophilin A, via its effects on the expression of CD 47 and calreticulin, impairs efferocytosis in apoptotic macrophages. Together with its impact on cholesterol efflux from macrophages, these effects can amplify other mechanisms of Cyp A in accelerating the progression of atherosclerosis.

Apoptotic cells generated during development and for tissue homeostasis are swiftly and continuously removed by phagocytes via a process called efferocytosis. Efficient efferocytosis can be achieved via transcriptional modulation in phagocytes that have engulfed apoptotic cells. However, such modulation and its effect on efferocytosis are not completely understood. Here, we report that phagocytes are recruited to apoptotic cells being cleared through the Mcp-1–Ccr2 axis, which facilitates clearance of apoptotic cells. We identified Mcp-1 as a modulated transcript using a microarray and found that Mcp-1 secretion was augmented in phagocytes engulfing apoptotic cells. This augmented Mcp-1 secretion was impaired by blocking phagolysosomal degradation of apoptotic cells. Conditioned medium from wild type (WT) phagocytes promoted cell migration, but that from Mcp-1−/− phagocytes did not. In addition, blockade of Ccr2, the receptor for Mcp-1, abrogated cell migration to conditioned medium from phagocytes incubated with apoptotic cells. The intrinsic efferocytosis activity of Mcp-1−/− and Ccr2−/− phagocytes was unaltered, but clearance of apoptotic cells was less efficient in the peritoneum of Mcp-1−/− and Ccr2−/− mice than in that of WT mice because fewer Ccr2-positive phagocytes were recruited. Taken together, our findings demonstrate a mechanism by which not only apoptotic cells but also phagocytes induce chemoattraction to recruit phagocytes to sites where apoptotic cells are cleared for efficient efferocytosis.

Efferocytosis, i.e., engulfment of dead cells by macrophages, is a crucial step during tissue repair after an injury. Efferocytosis delineates the transition from the pro-inflammatory phase of the inflammatory response to the recovery phase that ensures tissue reconstruction. We present here the role of efferocytosis during skeletal muscle regeneration, which is a paradigm of sterile tissue injury followed by a complete regeneration. We present the molecular mechanisms that have been described to control this process, and particularly the metabolic control of efferocytosis during skeletal muscle regeneration.

Swift and continuous phagocytosis of apoptotic cells can be achieved by modulation of calcium flux in phagocytes. However, the molecular mechanism by which apoptotic cells modulate calcium flux in phagocytes is incompletely understood. Here, using biophysical, biochemical, pharmaceutical, and genetic approaches, we show that apoptotic cells induced the Orai1-STIM1 interaction, leading to store-operated calcium entry (SOCE) in phagocytes through the Mertk-phospholipase C (PLC) γ1-inositol 1,4,5-triphosphate receptor (IP3R) axis. Apoptotic cells induced calcium release from the endoplasmic reticulum, which led to the Orai1-STIM1 association and, consequently, SOCE in phagocytes. This association was attenuated by masking phosphatidylserine. In addition, the depletion of Mertk, which indirectly senses phosphatidylserine on apoptotic cells, reduced the phosphorylation levels of PLCγ1 and IP3R, resulting in attenuation of the Orai1-STIM1 interaction and inefficient SOCE upon apoptotic cell stimulation. Taken together, our observations uncover the mechanism of how phagocytes engulfing apoptotic cells elevate the calcium level.

Following myocardial infarction (MI), blood neutrophils quickly and extensively infiltrate the heart, where they are temporally polarized into pro-inflammatory (N1) and anti-inflammatory (N2) subpopulations. Neutrophil transmigration is rapidly followed by the accrual of macrophages (MACs), which are believed to undergo local phenotypic transformations from pro-inflammatory to pro-healing MACs that mediate inflammation resolution. We hypothesized that N2 neutrophils can reprogram MACs toward a healing phenotype with increased efferocytosis capacity. To examine this, human neutrophils isolated from healthy subjects were polarized in N1 and N2 neutrophils, and their secretome was added to human MACs derived from THP monocytes. The impact of neutrophil factors on macrophages was investigated using qPCR, ELISA, Western blot, immunofluorescence, or an efferocytosis assay. The results show that the MACs exposed to N2 neutrophil secretome exhibited (i) increased expression of the anti-inflammatory molecules CD206 , TGF-β, and IL-10 and the nuclear factors associated with reparatory macrophages (PPARγ, Nur77, and KLF4 ); (ii) enhanced expression of efferocytosis receptors (MerTK, CD36 , CX3CR1, and integrins αv/β5 ) and of the bridge molecules Mfage8 and Gas6; and (iii) enhanced efferocytosis. In conclusion, factors released by N2 neutrophils induce a pro-healing phenotype of MACs by upregulating anti-inflammatory molecules and efferocytosis receptors and ensuing the efferocytosis capacity. The data suggest that molecular therapy to foster N2 polarization, which boosts macrophages' pro-healing phenotype, could be a promising strategy to speed up inflammation resolution and tissue repair.

Throughout oogenesis, Drosophila egg chambers traverse the fine line between survival and death. After surviving the ten early and middle stages of oogenesis, egg chambers drastically change their size and structure to produce fully developed oocytes. The development of an oocyte comes at a cost, the price is the lives of the oocyte’s 15 siblings, the nurse cells. These nurse cells do not die of their own accord. Their death is dependent upon their neighbors—the stretch follicle cells. Stretch follicle cells are nonprofessional phagocytes that spend the final stages of oogenesis surrounding the nurse cells and subsequently forcing the nurse cells to give up everything for the sake of the oocyte. In this review, we provide an overview of cell death in the ovary, with a focus on recent findings concerning this phagocyte-dependent non-autonomous cell death.

Apoptosis, the programmed and intentional death of senescent, damaged, or otherwise superfluous cells, is the natural end-point for most cells within multicellular organisms. Apoptotic cells are not inherently damaging, but if left unattended, they can lyse through secondary necrosis. The resulting release of intracellular contents drives inflammation in the surrounding tissue and can lead to autoimmunity. These negative consequences of secondary necrosis are avoided by efferocytosis—the phagocytic clearance of apoptotic cells. Efferocytosis is a product of both apoptotic cells and efferocyte mechanisms, which cooperate to ensure the rapid and complete removal of apoptotic cells. Herein, we review the processes used by apoptotic cells to ensure their timely removal, and the receptors, signaling, and cellular processes used by efferocytes for efferocytosis, with a focus on the receptors and signaling driving this process.

Tim-4 promotes the engulfment of apoptotic cells or exogenous particles by securing them on phagocytes. It is unable to transduce signals by itself but helps other engulfment receptors sense and internalize them. However, the identity of the engulfment receptors collaborating with Tim-4 is still incompletely understood. In this study, we searched for a candidate transmembrane protein with a FN3 domain, important for interaction with Tim-4, in silico and investigated whether it indeed interacts with Tim-4 and is involved in Tim-4-mediated phagocytosis. We found that EphA2 containing a FN3 domain in the extracellular region interacted with Tim-4, which was mediated by the IgV domain of Tim-4 and the FN3 domain of EphA2. Nevertheless, we found that EphA2 expression failed to alter Tim-4-mediated phagocytosis of apoptotic cells or polystyrene beads. Taken together, our findings suggest that EphA2, a new Tim-4 interacting protein, may intervene in a Tim-4-mediated cellular event even if it is not phagocytosis of endogenous or exogenous particles and vice versa.

Neonatal brain hypoxia-ischemia (HI) is a leading cause of morbidity and long-term disabilities in children. While we have made significant progress in describing HI mechanisms, the limited therapies currently offered for HI treatment in the clinical setting stress the importance of discovering new targetable pathways. Efferocytosis is an immunoregulatory and homeostatic process of clearance of apoptotic cells (AC) and cellular debris, best described in the brain during neurodevelopment. The therapeutic potential of stimulating defective efferocytosis has been recognized in neurodegenerative diseases. In this review, we will explore the involvement of efferocytosis after a stroke and HI as a promising target for new HI therapies.

Heme oxygenase-1 (HO-1) plays a vital role in the catabolism of heme and yields equimolar amounts of biliverdin, carbon monoxide, and free iron. We report that macrophages engulfing either the low amount of heme-containing apoptotic thymocytes or the high amount of heme-containing eryptotic red blood cells (eRBCs) strongly upregulate HO-1. The induction by apoptotic thymocytes is dependent on soluble signals, which do not include adenylate cyclase activators but induce the p38 mitogen-activated protein (MAP) kinase pathway, while in the case of eRBCs, it is cell uptake-dependent. Both pathways might involve the regulation of BTB and CNC homology 1 (BACH1), which is the repressor transcription regulator factor of the HO-1 gene. Long-term continuous efferocytosis of apoptotic thymocytes is not affected by the loss of HO-1, but that of eRBCs is inhibited. This latter is related to an internal signaling pathway that prevents the efferocytosis-induced increase in Rac1 activity. While the uptake of apoptotic cells suppressed the basal pro-inflammatory cytokine production in wild-type macrophages, in the absence of HO-1, engulfing macrophages produced enhanced amounts of pro-inflammatory cytokines. Our data demonstrate that HO-1 is required for both the engulfment and the anti-inflammatory response parts of the efferocytosis program.

The signal transducer and activator of transcription 6 (STAT6) transcription factor promotes activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway in macrophages. Little is known about the effect of proximal signal transduction leading to PPARγ activation for the resolution of acute inflammation. Here, we studied the role of STAT6 signaling in PPARγ activation and the resolution of acute sterile inflammation in a murine model of zymosan-induced peritonitis. First, we showed that STAT6 is aberrantly activated in peritoneal macrophages after zymosan injection. Utilizing STAT6−/− and wild-type (WT) mice, we found that STAT6 deficiency further enhanced zymosan-induced proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and macrophage inflammatory protein-2 in peritoneal lavage fluid (PLF) and serum, neutrophil numbers and total protein amount in PLF, but reduced proresolving molecules, such as IL-10 and hepatocyte growth factor, in PLF. The peritoneal macrophages and spleens of STAT6−/− mice exhibited lower mRNA and protein levels of PPARγ and its target molecules over the course of inflammation than those of WT mice. The deficiency of STAT6 was shown to impair efferocytosis by peritoneal macrophages. Taken together, these results suggest that enhanced STAT6 signaling results in PPARγ-mediated macrophage programming, contributing to increased efferocytosis and inflammation resolution.

The physiological fate of cells that die by apoptosis is their prompt and efficient removal by efferocytosis. During these processes, apoptotic cells release intracellular constituents that include purine nucleotides, lysophosphatidylcholine (LPC), and Sphingosine-1-phosphate (S1P) that induce migration and chemo-attraction of phagocytes as well as mitogens and extracellular membrane-bound vesicles that contribute to apoptosis-induced compensatory proliferation and alteration of the extracellular matrix and the vascular network. Additionally, during efferocytosis, phagocytic cells produce a number of anti-inflammatory and resolving factors, and, together with apoptotic cells, efferocytic events have a homeostatic function that regulates tissue repair. These homeostatic functions are dysregulated in cancers, where, aforementioned events, if not properly controlled, can lead to cancer progression and immune escape. Here, we summarize evidence that apoptosis and efferocytosis are exploited in cancer, as well as discuss current translation and clinical efforts to harness signals from dying cells into therapeutic strategies.

Apoptotic cells expressing phosphatidylserine (PS) on their cell surface are directly or indirectly recognized by phagocytes through PS-binding proteins. The PS-binding protein Tim-4 secures apoptotic cells to phagocytes to facilitate the engulfment of apoptotic cells. However, the molecular mechanism by which Tim-4 transduces signals to phagocytes during Tim-4-mediated efferocytosis is incompletely understood. Here, we report that Tim-4 collaborates with Mertk during efferocytosis through a biochemical interaction with Mertk. Proximal localization between the two proteins in phagocytes was observed by immunofluorescence and proximal ligation assays. Physical association between Tim-4 and Mertk, which was mediated by an interaction between the IgV domain of Tim-4 and the fibronectin type-III domain of Mertk, was also detected with immunoprecipitation. Furthermore, the effect of Mertk on Tim-4-mediated efferocytosis was abolished by GST-MertkFnIII, a soluble form of the fibronectin type-III domain of Mertk that disrupts the interaction between Tim-4 and Mertk. Taken together, the results from our study suggest that a physical interaction between Tim-4 and Mertk is necessary for Mertk to enhance efferocytosis mediated by Tim-4.

The clearance of apoptotic cells by macrophages (efferocytosis) is crucial to maintain normal tissue homeostasis; however, efferocytosis of cancer cells frequently results in inflammation and immunosuppression. Recently, we demonstrated that efferocytosis of apoptotic prostate cancer cells by bone marrow-derived macrophages induced a pro-inflammatory response that accelerated metastatic tumor growth in bone. To evaluate the microenvironmental impact of macrophages and their efferocytic function, we compared peritoneal macrophages (P-MΦ) versus bone marrow-derived macrophages (BM-MΦs) using an efferocytosis in vitro model. The capability to engulf apoptotic prostate cells was similar in BM-MΦs and P-MΦs. Ex vivo analysis of BM-MΦs showed an M2-like phenotype compared with a predominantly M1-like phenotype in P-MΦs. A distinct gene and protein expression profile of pro-inflammatory cytokines was found in BM-MΦs as compared with P-MΦs engulfing apoptotic prostate cancer cells. Importantly, the reprogramming of BM-MΦs toward an M1-like phenotype mitigated their inflammatory cytokine expression profile. In conclusion, BM-MΦs and P-MΦs are both capable of efferocytosing apoptotic prostate cancer cells; however, BM-MΦs exert increased inflammatory cytokine expression that is dependent upon the M2 polarization stage of macrophages. These findings suggest that bone marrow macrophage efferocytosis of apoptotic cancer cells maintains a unique pro-inflammatory microenvironment that may support a fertile niche for cancer growth. Finally, bone marrow macrophage reprogramming towards M1-type by interferon-γ (IFN-γ) induced a significant reduction in the efferocytosis-mediated pro-inflammatory signature.

Macrophages are central to inflammation resolution, an active process aimed at restoring tissue homeostasis following an inflammatory response. Here, the effects of db-cAMP on macrophage phenotype and function were investigated. Injection of db-cAMP into the pleural cavity of mice induced monocytes recruitment in a manner dependent on PKA and CCR2/CCL2 pathways. Furthermore, db-cAMP promoted reprogramming of bone-marrow-derived macrophages to a M2 phenotype as seen by increased Arg-1/CD206/Ym-1 expression and IL-10 levels (M2 markers). Db-cAMP also showed a synergistic effect with IL-4 in inducing STAT-3 phosphorylation and Arg-1 expression. Importantly, db-cAMP prevented IFN-γ/LPS-induced macrophage polarization to M1-like as shown by increased Arg-1 associated to lower levels of M1 cytokines (TNF-α/IL-6) and p-STAT1. In vivo, db-cAMP reduced the number of M1 macrophages induced by LPS injection without changes in M2 and Mres numbers. Moreover, db-cAMP enhanced efferocytosis of apoptotic neutrophils in a PKA-dependent manner and increased the expression of Annexin A1 and CD36, two molecules associated with efferocytosis. Finally, inhibition of endogenous PKA during LPS-induced pleurisy impaired the physiological resolution of inflammation. Taken together, the results suggest that cAMP is involved in the major functions of macrophages, such as nonphlogistic recruitment, reprogramming and efferocytosis, all key processes for inflammation resolution.

Uncontrolled inflammation leads to tissue damage and it is central for the development of chronic inflammatory diseases and autoimmunity. An acute inflammatory response is finely regulated by the action of anti-inflammatory and pro-resolutive mediators, culminating in the resolution of inflammation and restoration of homeostasis. There are few studies investigating intracellular signaling pathways associated with the resolution of inflammation. Here, we investigate the role of Rho-associated kinase (ROCK), a serine/threonine kinase, in a model of self-resolving neutrophilic inflammatory. We show that ROCK activity, evaluated by P-MYPT-1 kinetics, was higher during the peak of lipopolysaccharide-induced neutrophil influx in the pleural cavity of mice. ROCK inhibition by treatment with Y-27632 decreased the accumulation of neutrophils in the pleural cavity and was associated with an increase in apoptotic events and efferocytosis, as evaluated by an in vivo assay. In a model of gout, treatment with Y-27632 reduced neutrophil accumulation, IL-1β levels and hypernociception in the joint. These were associated with reduced MYPT and IκBα phosphorylation levels and increased apoptosis. Finally, inhibition of ROCK activity also induced apoptosis in human neutrophils and destabilized cytoskeleton, extending the observed effects to human cells. Taken together, these data show that inhibition of the ROCK pathway might represent a potential therapeutic target for neutrophilic inflammatory diseases.

Apoptosis and subsequent removal of dead cells are an essential part of wound healing. Macrophages phagocytize apoptotic cells (efferocytosis) and contribute to the resolution of inflammation. However, their participation in fibrogenesis and the mechanisms of influence on this process remain unclear. In the present study, we focused on the fibrogenic properties of human monocyte-derived macrophages polarized in the M2 direction by interaction with apoptotic cells. We studied their influence on the proliferation ([3H]-thymidine incorporation), differentiation (by the expression of α-SMA, a myofibroblast marker) and collagen-producing activity (ELISA) of dermal fibroblasts compared to classically (LPS) and alternatively (IL-4) activated macrophages. Macrophages polarized by the interaction with apoptotic cells had a unique phenotype and profile of produced factors and differed from the compared macrophage subtypes. Their conditioned media promoted the proliferation of dermal fibroblasts and the expression of α-SMA in them at the level of macrophages stimulated by IL-4, while the stimulating effect on the collagen-producing activity was more pronounced compared to that of the other macrophage subtypes. Moreover, they are characterized by the high level of production of pro-fibrotic factors such as TIMP-1, TGF-β1 and angiogenin. Taken together, M2-like macrophages polarized by efferocytosis demonstrate in vitro pro-fibrotic activity by promoting the functional activity of dermal fibroblasts and producing pro-fibrotic and pro-angiogenic factors.

Neutrophils are highly abundant circulating leukocytes that are amongst the first cells to be recruited to sites of infection or sterile injury. Their ability to generate and release powerful cytotoxic products ties with their role in host defence from bacterial and fungal infections. Neutrophilic inflammation is tightly regulated to limit the amount of 'bystander injury' caused. Neutrophils were in the past regarded as short-lived, indiscriminate killers of invading microorganisms. However, this view has changed quite dramatically in recent years. Amongst other insights, neutrophils are now recognised to also have important anti-inflammatory functions that are critical for the resolution of inflammation and return to homeostasis. This minireview focusses on anti-inflammatory neutrophil functions, placing a particular focus on recent findings linked to neutrophil cell death, several types of which may be anti-inflammatory (apoptosis, secondary necrosis, and neutrophil extracellular traps). These are discussed together with features that may further promote the clearance of dead cells by efferocytosis and reprogramming of macrophages to promote resolution and repair.

The clearance of apoptotic cancer cells by macrophages, known as efferocytosis, fuels the bone-metastatic growth of prostate cancer cells via pro-inflammatory and immunosuppressive processes. However, the exact molecular mechanisms remain unclear. In this study, single-cell transcriptomics of bone marrow (BM) macrophages undergoing efferocytosis of apoptotic prostate cancer cells revealed a significant enrichment in their cellular response to hypoxia. Here, we show that BM macrophage efferocytosis increased hypoxia inducible factor-1alpha (HIF-1α) and STAT3 phosphorylation (p-STAT3 at Tyr705) under normoxic conditions, while inhibitors of p-STAT3 reduced HIF-1α. Efferocytosis promoted HIF-1α stabilization, reduced its ubiquitination, and induced HIF-1α and p-STAT3 nuclear translocation. HIF-1α stabilization in efferocytic BM macrophages resulted in enhanced expression of pro-inflammatory cytokine MIF, whereas BM macrophages with inactive HIF-1α reduced MIF expression upon efferocytosis. Stabilization of HIF-1α using the HIF-prolyl-hydroxylase inhibitor, Roxadustat, enhanced MIF expression in BM macrophages. Furthermore, BM macrophages treated with recombinant MIF protein activated NF-κB (p65) signaling and increased the expression of pro-inflammatory cytokines. Altogether, these findings suggest that the clearance of apoptotic cancer cells by BM macrophages triggers p-STAT3/HIF-1α/MIF signaling to promote further inflammation in the bone tumor microenvironment where a significant number of apoptotic cancer cells are present.

Every day, billions of our cells die and get cleared without inducing inflammation. When, clearance is improper, uncleared cells undergo secondary necrosis and trigger inflammation. In addition, proper efferocytosis would be required for inducing resolution of inflammation, thus clearance deficiencies in the long term lead to development of various chronic inflammatory diseases. Increasing evidence indicates that obesity, itself being a low-grade inflammatory disease, predisposes to a variety of other chronic inflammatory diseases. Previous studies indicated that this later might be partially related to an impaired efferocytosis induced by increased uptake of circulating saturated fatty acids by macrophages in obese people. Here, we show that palmitate inhibits efferocytosis by bone marrow-derived macrophages in a dose-dependent manner. Palmitate triggers autophagy but also activates an energy-sensing mTORC1/ROCK1 signaling pathway, which interferes with the autophagosome-lysosome fusion, resulting in accumulation of the cellular membranes in autophagosomes. We propose that lack of sufficient plasma membrane supply attenuates efferocytosis of palmitate-exposed macrophages. AMP-activated protein kinase activators lead to mTORC1 inhibition and, consequently, released the palmitate-induced efferocytosis block in macrophages. Thus, they might be useful in the treatment of obesity not only by affecting metabolism thought so far. ROCK1 inhibitors could also be considered., Competing Interests: The authors declare no conflict of interest.

Clearance of apoptotic cells by bone marrow-derived macrophages differentiated from monocytes plays a central role in the resolution of inflammation, as the conversion of pro-inflammatory M1 macrophages to M2 macrophages that mediate the resolution process occurs during efferocytosis. Thus, proper efferocytosis is a prerequisite for proper resolution of inflammation, and failure in efferocytosis is associated with the development of chronic inflammatory diseases. Previous studies from our laboratory have shown that (13 R )-all- trans -13,14-dihydroretinol (DHR), the product of retinol saturase, acting from day 4 of monocyte differentiation enhances the efferocytosis capacity of the resulted macrophages. Loss of retinol saturase in mice leads to impaired efferocytosis, and to development of autoimmunity. In the present paper, we report that in differentiating monocytes DHR, retinol, and all- trans retinoic acid all act directly on retinoic acid receptors and enhance the clearance of apoptotic cells by upregulating the expression of several efferocytosis-related genes. The effect of retinoids seems to be mediated by bone morphogenetic protein (BMP)-2, and the Smad3 transcription factor. In addition, retinoids also upregulate the expression of the vitamin D receptor and that of vascular endothelial growth factor A, indicating that altogether retinoids promote the generation of a pro-reparative M2 macrophage population during monocyte differentiation.

Mesenchymal stromal cells (MSCs) are largely known for their immune-suppressive capacity, hence, their common use in the control of unwanted inflammation. However, novel concepts related to their biology, combined with the urgent need to identify MSC subpopulations with enhanced suppressive properties, drive the search for isolation protocols optimized for clinical applications. We show, in this study, that MSCs expressing high CD146 levels exhibit altered surface expression profiles of CD44 and secrete elevated levels of interleukin (IL)-6, amongst other factors. In addition, CD146 hi MSCs surpass the polyclonal parental populations in inhibiting alloreactive T cells in vitro, in both a soluble- and cell-contact-dependent manner. Despite the lack of CD146 hi MSC-mediated activation of peritoneal macrophages to release the suppressive factor IL-10 in vitro, their administration in animals with graft-versus-host disease alleviates inflammation and leads to 40% survival rate up to 7 weeks post-transplantation. This pronounced inhibitory property is driven by CD146-mediated in situ efferocytosis by myeloid cells. Altogether, this study provides the impetus to adopt an isolation protocol for MSCs based on a CD146 expression profile before their therapeutic use and suggests a major role played by CD146 as a novel "eat-me" signal, capable of enhancing MSC uptake by competent phagocytes.

The extensive use of mesenchymal stromal cells (MSCs) over the last decade has revolutionized modern medicine. From the delivery of pharmacological proteins to regenerative medicine and immune modulation, these cells have proven to be highly pleiotropic and responsive to their surrounding environment. Nevertheless, their role in promoting inflammation has been fairly limited by the questionable use of interferon-gamma, as this approach has also been proven to enhance the cells' immune-suppressive abilities. Alternatively, we have previously shown that de novo expression of the immunoproteasome (IPr) complex instills potent antigen cross-presentation capabilities in MSCs. Interestingly, these cells were found to express the major histocompatibility class (MHC) II protein, which prompted us to investigate their ability to stimulate humoral immunity. Using a series of in vivo studies, we found that administration of allogeneic ovalbumin (OVA)-pulsed MSC-IPr cells elicits a moderate antibody titer, which was further enhanced by the combined use of pro-inflammatory cytokines. The generated antibodies were functional as they blocked CD4 T-cell activation following their co-culture with OVA-pulsed MSC-IPr and mitigated E.G7 tumor growth in vivo. The therapeutic potency of MSC-IPr was, however, dependent on efferocytosis, as phagocyte depletion prior to vaccination abrogated MSC-IPr-induced humoral responses while promoting their survival in the host. In contrast, antibody-mediated neutralization of CD47, a potent "do not eat me signal", enhanced antibody titer levels. These observations highlight the major role played by myeloid cells in supporting antibody production by MSC-IPr and suggest that the immune outcome is dictated by a net balance between efferocytosis-stimulating and -inhibiting signals.

Impairment of efferocytosis in apoptotic macrophages is a known determinant of the severity of atherosclerosis and the vulnerability of plaques to rupture. The precise mechanisms involved in impaired efferocytosis are unclear. Given the well-recognized role of the inflammatory cytokine cyclophilin A (Cyp A) in modulating several atherogenic mechanisms in high-glucose primed monocytes, we investigated the role of Cyp A in macrophage efferocytosis. The efficiency of efferocytosis in RAW 264.7 macrophages grown in vitro and primed with cyclophilin A was assessed using flow cytometry and confocal assays. Cholesterol content in cells was measured using cell-based cholesterol efflux assay. Proteomic analysis and bioinformatics tools were employed to decipher the link between cyclophilin A and the known ligand receptors involved in efferocytosis. Cyclophilin A was found to impair efferocytosis in apoptotic macrophages by reducing ABCA1-mediated cholesterol efflux in foam cells derived from macrophages. Cyclophilin A-primed macrophages showed an increase in expression of the don't-eat-me signal CD 47 and a decrease in the expression of the eat-me signal, calreticulin. Phagocytosis was restored upon silencing of cyclophilin A. New Zealand white rabbits were fed a high-fat diet, and lesions in their aortae were analyzed histologically for evidence of atherosclerosis and the expression of Cyp A, CD 47 and calreticulin, the ligand receptor involved in efferocytosis. Gene and protein expressions in aortae and macrophages were analyzed by real-time PCR and Western blotting. Cyclophilin A, via its effects on the expression of CD 47 and calreticulin, impairs efferocytosis in apoptotic macrophages. Together with its impact on cholesterol efflux from macrophages, these effects can amplify other mechanisms of Cyp A in accelerating the progression of atherosclerosis.

Efferocytosis, i.e., engulfment of dead cells by macrophages, is a crucial step during tissue repair after an injury. Efferocytosis delineates the transition from the pro-inflammatory phase of the inflammatory response to the recovery phase that ensures tissue reconstruction. We present here the role of efferocytosis during skeletal muscle regeneration, which is a paradigm of sterile tissue injury followed by a complete regeneration. We present the molecular mechanisms that have been described to control this process, and particularly the metabolic control of efferocytosis during skeletal muscle regeneration.

Apoptotic cells generated during development and for tissue homeostasis are swiftly and continuously removed by phagocytes via a process called efferocytosis. Efficient efferocytosis can be achieved via transcriptional modulation in phagocytes that have engulfed apoptotic cells. However, such modulation and its effect on efferocytosis are not completely understood. Here, we report that phagocytes are recruited to apoptotic cells being cleared through the Mcp-1-Ccr2 axis, which facilitates clearance of apoptotic cells. We identified Mcp-1 as a modulated transcript using a microarray and found that Mcp-1 secretion was augmented in phagocytes engulfing apoptotic cells. This augmented Mcp-1 secretion was impaired by blocking phagolysosomal degradation of apoptotic cells. Conditioned medium from wild type (WT) phagocytes promoted cell migration, but that from Mcp-1 -/- phagocytes did not. In addition, blockade of Ccr2, the receptor for Mcp-1, abrogated cell migration to conditioned medium from phagocytes incubated with apoptotic cells. The intrinsic efferocytosis activity of Mcp-1 - / - phagocytes was unaltered, but clearance of apoptotic cells was less efficient in the peritoneum of Ccr2 - / - phagocytes was unaltered, but clearance of apoptotic cells was less efficient in the peritoneum of Mcp-1 - mice because fewer Ccr2-positive phagocytes were recruited. Taken together, our findings demonstrate a mechanism by which not only apoptotic cells but also phagocytes induce chemoattraction to recruit phagocytes to sites where apoptotic cells are cleared for efficient efferocytosis./ - and Ccr2 - / - mice than in that of WT mice because fewer Ccr2-positive phagocytes were recruited. Taken together, our findings demonstrate a mechanism by which not only apoptotic cells but also phagocytes induce chemoattraction to recruit phagocytes to sites where apoptotic cells are cleared for efficient efferocytosis.

Swift and continuous phagocytosis of apoptotic cells can be achieved by modulation of calcium flux in phagocytes. However, the molecular mechanism by which apoptotic cells modulate calcium flux in phagocytes is incompletely understood. Here, using biophysical, biochemical, pharmaceutical, and genetic approaches, we show that apoptotic cells induced the Orai1-STIM1 interaction, leading to store-operated calcium entry (SOCE) in phagocytes through the Mertk-phospholipase C (PLC) γ1-inositol 1,4,5-triphosphate receptor (IP 3 R) axis. Apoptotic cells induced calcium release from the endoplasmic reticulum, which led to the Orai1-STIM1 association and, consequently, SOCE in phagocytes. This association was attenuated by masking phosphatidylserine. In addition, the depletion of Mertk, which indirectly senses phosphatidylserine on apoptotic cells, reduced the phosphorylation levels of PLCγ1 and IP 3 R, resulting in attenuation of the Orai1-STIM1 interaction and inefficient SOCE upon apoptotic cell stimulation. Taken together, our observations uncover the mechanism of how phagocytes engulfing apoptotic cells elevate the calcium level.

The safe removal of apoptotic debris by macrophages-often referred to as efferocytosis-is crucial for maintaining tissue integrity and preventing self-immunity or tissue damaging inflammation. Macrophages clear tissues of hazardous materials from dying cells and ultimately adopt a pro-resolving activation state. However, adipocyte apoptosis is an inflammation-generating process, and the removal of apoptotic adipocytes by so-called adipose tissue macrophages triggers a sequence of events that lead to meta-inflammation and obesity-associated metabolic diseases. Signals that allow apoptotic cells to control macrophage immune functions are complex and involve metabolites released by the apoptotic cells and also metabolites produced by the macrophages during the digestion of apoptotic cell contents. This review provides a concise summary of the adipocyte-derived metabolites that potentially control adipose tissue macrophage immune functions and, hence, may induce or alleviate adipose tissue inflammation.

Throughout oogenesis, Drosophila egg chambers traverse the fine line between survival and death. After surviving the ten early and middle stages of oogenesis, egg chambers drastically change their size and structure to produce fully developed oocytes. The development of an oocyte comes at a cost, the price is the lives of the oocyte's 15 siblings, the nurse cells. These nurse cells do not die of their own accord. Their death is dependent upon their neighbors-the stretch follicle cells. Stretch follicle cells are nonprofessional phagocytes that spend the final stages of oogenesis surrounding the nurse cells and subsequently forcing the nurse cells to give up everything for the sake of the oocyte. In this review, we provide an overview of cell death in the ovary, with a focus on recent findings concerning this phagocyte-dependent non-autonomous cell death.

Tim-4 promotes the engulfment of apoptotic cells or exogenous particles by securing them on phagocytes. It is unable to transduce signals by itself but helps other engulfment receptors sense and internalize them. However, the identity of the engulfment receptors collaborating with Tim-4 is still incompletely understood. In this study, we searched for a candidate transmembrane protein with a FN3 domain, important for interaction with Tim-4, in silico and investigated whether it indeed interacts with Tim-4 and is involved in Tim-4-mediated phagocytosis. We found that EphA2 containing a FN3 domain in the extracellular region interacted with Tim-4, which was mediated by the IgV domain of Tim-4 and the FN3 domain of EphA2. Nevertheless, we found that EphA2 expression failed to alter Tim-4-mediated phagocytosis of apoptotic cells or polystyrene beads. Taken together, our findings suggest that EphA2, a new Tim-4 interacting protein, may intervene in a Tim-4-mediated cellular event even if it is not phagocytosis of endogenous or exogenous particles and vice versa.

Apoptosis, the programmed and intentional death of senescent, damaged, or otherwise superfluous cells, is the natural end-point for most cells within multicellular organisms. Apoptotic cells are not inherently damaging, but if left unattended, they can lyse through secondary necrosis. The resulting release of intracellular contents drives inflammation in the surrounding tissue and can lead to autoimmunity. These negative consequences of secondary necrosis are avoided by efferocytosis-the phagocytic clearance of apoptotic cells. Efferocytosis is a product of both apoptotic cells and efferocyte mechanisms, which cooperate to ensure the rapid and complete removal of apoptotic cells. Herein, we review the processes used by apoptotic cells to ensure their timely removal, and the receptors, signaling, and cellular processes used by efferocytes for efferocytosis, with a focus on the receptors and signaling driving this process.

The signal transducer and activator of transcription 6 (STAT6) transcription factor promotes activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway in macrophages. Little is known about the effect of proximal signal transduction leading to PPARγ activation for the resolution of acute inflammation. Here, we studied the role of STAT6 signaling in PPARγ activation and the resolution of acute sterile inflammation in a murine model of zymosan-induced peritonitis. First, we showed that STAT6 is aberrantly activated in peritoneal macrophages after zymosan injection. Utilizing STAT6 -/- and wild-type (WT) mice, we found that STAT6 deficiency further enhanced zymosan-induced proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and macrophage inflammatory protein-2 in peritoneal lavage fluid (PLF) and serum, neutrophil numbers and total protein amount in PLF, but reduced proresolving molecules, such as IL-10 and hepatocyte growth factor, in PLF. The peritoneal macrophages and spleens of STAT6 -/- mice exhibited lower mRNA and protein levels of PPARγ and its target molecules over the course of inflammation than those of WT mice. The deficiency of STAT6 was shown to impair efferocytosis by peritoneal macrophages. Taken together, these results suggest that enhanced STAT6 signaling results in PPARγ-mediated macrophage programming, contributing to increased efferocytosis and inflammation resolution.