Efferocytosis is the process by which phagocytes recognize, engulf, and degrade dying cells before they undergo secondary necrosis. Clearance of apoptotic cells is the most typical form of efferocytosis and involves phagocyte recruitment, phosphatidylserine recognition, phagocytic receptor activation, actin-cytoskeleton remodeling, phagosome maturation, lysosomal degradation, and immunometabolic adaptation. It is an important mechanism for maintaining tissue homeostasis, limiting the spread of inflammation, and promoting tissue repair.

Keywords: efferocytosis; apoptotic cells; phosphatidylserine; MerTK; CD47; SIRPα; Rac1; phagosome; LC3-associated phagocytosis; immunometabolism

1 Basic Characteristics of Efferocytosis

1.1 Definition of Efferocytosis

Efferocytosis mainly refers to the process by which macrophages and other phagocytic cells recognize, engulf, and clear apoptotic cells and apoptotic bodies. Its major function is to remove apoptotic cells before substantial plasma-membrane rupture occurs, thereby preventing secondary necrosis and the release of nucleic acids, proteases, and other damage-associated molecules. Efferocytosis can be summarized as a series of consecutive stages, including phagocyte recruitment, recognition and uptake of dying cells, phagosome maturation, and degradation of dying cells.

1.2 Major Effector Cells of Efferocytosis

Macrophages are the principal professional phagocytes responsible for efferocytosis in most tissues and possess strong capacities for dying-cell recognition, internalization, degradation, and continual engulfment. Dendritic cells and certain other myeloid cells can also participate in dying-cell clearance. Epithelial cells, endothelial cells, fibroblasts, and certain tissue parenchymal cells are nonprofessional phagocytes that mainly clear nearby dying cells. Their migration range and continual engulfment capacity are generally lower than those of professional phagocytes.

1.3 Physiological Significance of Efferocytosis

Large numbers of dying cells are continuously generated during tissue renewal. Effective efferocytosis creates space for newly generated cells, maintains stable tissue architecture and cell numbers, and limits damage caused by dying-cell contents to surrounding cells and the extracellular matrix. When the rate of dying-cell generation exceeds the clearance capacity of phagocytes, apoptotic cells may undergo secondary necrosis, release damage-associated molecular patterns, and increase autoantigen exposure, thereby promoting persistent inflammation and autoimmune responses.

1.4 Relationship Between Efferocytosis and Other Dying-Cell Clearance Processes

Classical efferocytosis mainly targets apoptotic cells whose plasma membranes remain relatively intact, whereas pyroptosis, necroptosis, and other lytic forms of cell death can directly disrupt the plasma membrane and release inflammatory contents. Although phagocytes also clear these dying cells, their recognition mechanisms and immunological consequences are not completely equivalent to those of classical efferocytosis. Phosphatidylserine externalization can also occur during multiple forms of cell death or cellular stress. Therefore, detection of phosphatidylserine alone cannot determine the form of cell death or demonstrate that the complete efferocytosis process has occurred.

Figure 1. Mechanisms of apoptotic cell recognition, phagosome maturation, and immune reprogramming during efferocytosis

2 Core Molecular Mechanisms of Efferocytosis

2.1 “Find-Me” Signals and Phagocyte Recruitment

Apoptotic cells can release nucleotides such as ATP and UTP, lipid mediators such as sphingosine-1-phosphate, and chemokines such as CX3CL1. These soluble signals can form local concentration gradients that guide monocytes, macrophages, and other migratory phagocytes toward the location of dying cells and regulate the expression of molecules associated with phagocytic receptors, the cytoskeleton, and intracellular digestive systems.

2.2 Pannexin 1 Channels and Nucleotide Release

During apoptosis, Caspase-3 and Caspase-7 can cleave and activate Pannexin 1 channels, promoting the release of ATP and UTP into the extracellular environment. After the released nucleotides bind purinergic receptors on phagocytes, they can regulate cell migration and phagocytic readiness. Extracellular ATP and AMP can also be further converted into adenosine, which suppresses certain pro-inflammatory signals and promotes inflammation-resolution-associated responses through corresponding receptors.

2.3 Phosphatidylserine Externalization

Phosphatidylserine (PS) is the most representative “Eat-Me” signal in efferocytosis. In healthy cells, PS is mainly distributed on the inner leaflet of the plasma membrane. After cells enter apoptosis, membrane-phospholipid asymmetry is disrupted and PS gradually becomes exposed on the cell surface, allowing phagocytes to recognize and clear apoptotic cells before they undergo secondary necrosis.

2.4 Direct Recognition of PS

Phagocytes can directly recognize PS on the surface of apoptotic cells through BAI1, TIM4, and other PS-binding receptors. After binding PS, BAI1 can recruit ELMO and DOCK proteins to form a signaling complex that promotes Rac1 activation. This transmits the recognition signal from the dying-cell surface into the phagocyte and initiates actin-cytoskeleton remodeling and formation of the phagocytic cup.

2.5 Indirect Recognition of PS

MerTK, AXL, and TYRO3 constitute the TAM receptor family. These receptors generally recognize PS indirectly through bridging molecules such as GAS6 or Protein S. One end of the bridging molecule binds PS on the apoptotic-cell surface, whereas the other end binds a TAM receptor on the phagocyte, thereby promoting receptor activation, cytoskeletal remodeling, and dying-cell uptake. MerTK is an important receptor for homeostatic apoptotic-cell clearance.

2.6 “Don’t-Eat-Me” Signals

Healthy cells can avoid inappropriate engulfment by expressing “Don’t-Eat-Me” signals such as CD47. After CD47 binds SIRPα on the phagocyte surface, it can induce phosphorylation of the SIRPα cytoplasmic domain and recruit SHP1 and SHP2, thereby inhibiting integrin activation, myosin contraction, and phagocytic-cup closure. Whether efferocytosis occurs depends on the integrated balance among pro-phagocytic signals, bridging molecules, phagocytic receptors, and inhibitory signals.

2.7 The ELMO-DOCK-Rac1 Signaling Axis

Dying-cell recognition signals can promote conversion of Rac1 from its GDP-bound state to its GTP-bound state through the ELMO-DOCK complex. Activated Rac1 subsequently regulates WAVE, Arp2/3, and other actin-regulatory components, promoting local F-actin polymerization and plasma-membrane extension. The phagocyte plasma membrane gradually extends around the dying cell to form a phagocytic cup, followed by membrane closure and complete internalization.

2.8 Phagosome Maturation and Lysosomal Fusion

After closure of the phagocytic cup, the dying cell is enclosed within a single-membrane structure to form a phagosome. Early phagosomes can recruit VPS34 and acquire markers such as RAB5. They subsequently transition into phagosomes with late-stage characteristics such as RAB7 and fuse with lysosomes through membrane-trafficking and SNARE-associated mechanisms to form phagolysosomes. The acidic environment, proteases, nucleases, and lipases within phagolysosomes degrade the components of dying cells.

2.9 LC3-Associated Phagocytosis

LC3-associated phagocytosis (LAP) is an important noncanonical autophagy mechanism that promotes phagosome maturation and dying-cell degradation. LAP occurs on single-membrane phagosomes containing extracellularly acquired cargo. VPS34, Beclin 1, UVRAG, Rubicon, and the LC3-conjugation system can participate in this process, allowing LC3 to be conjugated to the phagosomal membrane and promoting phagosome maturation and lysosomal fusion. LAP differs from classical double-membrane autophagy. Therefore, increased LC3 expression or punctate accumulation alone cannot demonstrate enhanced LAP.

3 Cellular and Biochemical Features of Efferocytosis

3.1 Phagocyte Migration

Under the influence of “Find-Me” signals, phagocytes may exhibit directional migration, extension of membrane protrusions, and accumulation around dying cells. Migration distance, migration speed, and the spatial distribution of phagocytes around dying cells can be used to evaluate the recruitment stage. However, arrival of phagocytes near dying cells and physical contact between the two cell types do not demonstrate that the dying cells have been internalized.

3.2 PS Externalization and Changes in Plasma-Membrane Integrity

Apoptotic target cells suitable for efferocytosis experiments are generally Annexin V-positive and remain PI- or 7-AAD-negative during the early stage. As apoptosis progresses and secondary necrosis occurs, plasma-membrane permeability increases, allowing PI or 7-AAD to enter the cells. If the proportion of target cells with lost membrane integrity is excessively high, released intracellular substances may directly activate phagocytes and interfere with analysis of classical efferocytosis.

3.3 Phagocytic Cups and Cytoskeletal Changes

After activation of phagocytic receptors, local F-actin polymerization occurs in phagocytes, and the plasma membrane gradually extends around dying cells to form ring-shaped or cup-shaped actin structures. When actin polymerization is inhibited, phagocytes may still bind dying cells but cannot complete phagocytic-cup closure and internalization. Therefore, adhesion of dying cells to phagocytes and true entry into the phagocyte interior need to be evaluated separately.

3.4 Phagosome Acidification and Dying-Cell Degradation

After dying cells are internalized, phagosomes gradually acquire characteristics of acidic compartments and become colocalized with lysosomal markers. Increased signals from acid-sensitive probes may indicate that dying cells have entered acidified phagosomes or phagolysosomes. However, acidification alone does not demonstrate complete degradation of dying cells. Attenuation of target-cell fluorescence, disappearance of cellular structures, and degradation of proteins or nucleic acids should also be observed.

3.5 Immune and Metabolic Changes

Efferocytosis is generally accompanied by increased anti-inflammatory factors such as IL-10 and decreased levels of certain pro-inflammatory factors. At the same time, phagocytes need to process cholesterol, fatty acids, amino acids, and nucleotides derived from dying cells and may therefore exhibit changes in cholesterol efflux, fatty acid oxidation, glycolysis, and amino acid metabolism. These indicators are functional outputs after efferocytosis and cannot replace direct evidence of dying-cell internalization and degradation.

4 Differences Between Efferocytosis and Related Cellular Clearance Processes

5 Experimental Models of Efferocytosis

5.1 Selection of Phagocyte Models

In vitro efferocytosis experiments can use primary macrophages, peripheral blood monocyte-derived macrophages, bone marrow-derived macrophages, or suitable macrophage cell lines. Dendritic cells, epithelial cells, or fibroblasts can also be used to investigate nonprofessional efferocytosis in specific tissues. Different cells vary in the expression of MerTK, AXL, TIM4, SIRPα, and other receptors. Therefore, receptor backgrounds should be defined before experiments, and cell-differentiation and culture conditions should be standardized.

5.2 Preparation of Apoptotic Target Cells

Jurkat T cells and other suspension cells are commonly used as apoptotic target cells in efferocytosis experiments and can be induced to undergo apoptosis using Staurosporine, ultraviolet irradiation, or other suitable methods. The induction conditions should produce stable PS externalization while preserving plasma-membrane integrity as much as possible and avoiding extensive secondary necrosis caused by excessively high treatment doses or prolonged exposure.

5.3 Fluorescent Labeling and Coculture

Phagocytes and apoptotic target cells can be labeled separately and cocultured at a defined ratio. The target-cell-to-phagocyte ratio, coculture duration, medium volume, serum conditions, and washing procedures should be standardized. Tracing dyes with good intracellular stability should be selected to avoid false-positive results caused by dye leakage or transfer from target cells to phagocytes.

5.4 Receptor- and Signaling-Intervention Models

Blocking antibodies, soluble receptors, kinase inhibitors, siRNA, or gene knockout can be used to intervene in molecules such as MerTK, AXL, CD47, SIRPα, ELMO, DOCK, and Rac1. After intervention, phagocyte migration, dying-cell binding, true internalization, and cell viability should be evaluated simultaneously to avoid misinterpreting reduced migration or drug-induced cytotoxicity as inhibition of the efferocytosis pathway.

5.5 Phagosome-Maturation and LAP-Intervention Models

Intervention in VPS34-associated signaling, inhibition of lysosomal acidification, LC3 detection, and knockdown of LAP-associated molecules can be used to analyze phagosome maturation and dying-cell degradation. Lysosomal-intervention agents such as Chloroquine can disrupt the endolysosomal system and block late-stage degradative flux, resulting in accumulation of phagosomes or LC3-associated structures. Such accumulation should not be interpreted as enhanced efferocytosis or LAP and may instead reflect blockade of late-stage degradation.

5.6 CharON Live-Cell Tracking Model

CharON is a reporter system that combines a Caspase-responsive green fluorescent module with a pH-sensitive red fluorescent module and can be used to continuously observe apoptosis, phagocyte recruitment, dying-cell uptake, and phagosome acidification. The green signal reflects apoptosis-associated Caspase activation, whereas the red signal increases as dying cells enter acidified compartments, thereby connecting the occurrence of cell death with subsequent clearance processes.

6 Experimental Identification of Efferocytosis

6.1 Confirmation of Apoptotic Target-Cell Status

Efferocytosis experiments first require confirmation that target cells are in an apoptotic state suitable for clearance. Annexin V positivity indicates PS externalization, while PI or 7-AAD is used to evaluate plasma-membrane integrity. Caspase-3/7 activity and nuclear morphology can provide additional support for identification of apoptosis. An ideal target-cell population should have a high proportion of Annexin V-positive cells while maintaining a controlled proportion of PI- or 7-AAD-positive cells.

6.2 Distinguishing Surface Binding From True Internalization

Detection of phagocyte and target-cell double positivity by flow cytometry only demonstrates contact, binding, or coexistence of fluorescent signals and does not directly prove that dying cells have entered the phagocyte interior. Confocal microscopic optical sectioning, three-dimensional reconstruction, extracellular fluorescence quenching, differential staining, or acid washing can be used to distinguish dying cells attached to the phagocyte surface from dying cells located within intracellular phagosomes.

6.3 Efferocytosis Efficiency and Continual Engulfment Capacity

Efferocytosis efficiency can be evaluated by measuring the proportion of phagocytes that have internalized at least one apoptotic cell, the number of dying cells within each phagocyte, target-cell fluorescence intensity within phagocytes, and the rate of dying-cell clearance per unit time. The proportion of positive phagocytes mainly reflects whether uptake has occurred, whereas the number of target cells within each phagocyte is more suitable for evaluating phagocytic load and continual efferocytosis capacity.

6.4 Validation of Phagosome Maturation and Degradation

Acidic-compartment probes, lysosomal labeling, attenuation of target-cell fluorescence, and changes in degradation products can be used to evaluate phagosome maturation. Colocalization of dying cells with probes such as LysoTracker supports entry into acidic compartments but cannot independently demonstrate complete degradation. Disappearance of target-cell structures, degradation of cellular contents, and subsequent clearance capacity of the phagocytes should also be evaluated.

6.5 Validation of Phagocytic Receptor Dependence

Intervention in MerTK, AXL, BAI1, TIM4, or the CD47-SIRPα pathway can be used to determine the roles of corresponding receptors in specific cellular models. Correlation between receptor expression and efferocytosis efficiency provides only associative evidence. Confirmation of causality requires receptor blockade, pharmacological inhibition, gene knockdown, or gene knockout, followed by observation of consistent changes in the efferocytosis phenotype after excluding cytotoxicity.

6.6 Validation of LAP Dependence

Identification of LAP requires demonstration that LC3 is recruited to single-membrane phagosomes containing dying cells and that this process affects phagosome maturation and dying-cell degradation. Increased LC3-II, punctate LC3 accumulation, or colocalization of LC3 with lysosomes cannot independently distinguish LAP from classical autophagy. Dying-cell localization, phagosome structure, intervention in LAP-associated molecules, and degradation function should be evaluated together.

6.7 Detection of Immune and Metabolic Outputs

IL-10, TNF-α, IL-1β, and other inflammatory factors can be measured to evaluate changes in immune status after efferocytosis. ABCA1, cholesterol, ATP, and energy-metabolism-associated indicators can also be detected to assess adaptation of phagocytes to the metabolic load imposed by dying cells. Changes in inflammatory factors and metabolic indicators should be interpreted only after true internalization of dying cells has been established and cannot independently identify efferocytosis.

7 Regulatory Factors of Efferocytosis

7.1 Balance Between “Eat-Me” and “Don’t-Eat-Me” Signals

Efferocytosis efficiency depends on the balance between pro-phagocytic signals such as PS and calreticulin and inhibitory signals such as CD47. Increased pro-phagocytic signals may not overcome strong CD47-SIRPα inhibition, whereas CD47 blockade cannot automatically trigger complete phagocytosis in the absence of effective pro-phagocytic signals. Therefore, different cell models may exhibit markedly different responses to the same intervention.

7.2 Dying-Cell Load and Continual Efferocytosis

The number of dying cells in tissues may exceed the number of phagocytes, and a single macrophage often needs to continually clear multiple apoptotic cells. An excessive dying-cell load can increase phagocyte volume, increase lysosomal burden, and reduce subsequent uptake capacity. Therefore, continual efferocytosis depends not only on restoration of the actin cytoskeleton but also on the timely processing of ingested lipids, proteins, and nucleic acids.

7.3 Cholesterol Processing and ABCA1

Apoptotic-cell membranes contain large amounts of cholesterol and phospholipids. Dying-cell uptake can promote ABCA1 expression and cholesterol efflux in phagocytes, thereby limiting lipid overload and maintaining continual clearance capacity. If cholesterol efflux is insufficient, lipid accumulation may occur in phagocytes and affect membrane structure, lysosomal function, and subsequent efferocytosis.

7.4 Fatty Acid Oxidation and IL-10 Expression

Lipids derived from dying cells can enter fatty acid metabolism in phagocytes, and changes in fatty acid oxidation and mitochondrial metabolism may be associated with IL-10 expression and inflammation-resolution functions. Changes in fatty acid oxidation should be analyzed together with dying-cell uptake, mitochondrial function, and cell-survival status and cannot independently demonstrate enhanced efferocytosis.

7.5 Inflammatory Environment and Phagocyte State

Pro-inflammatory cytokines, pathogen-associated signals, and oxidative stress can alter phagocytic receptor expression, cytoskeletal state, and lysosomal function. Macrophages in different activation states may exhibit substantial differences in migration, dying-cell recognition, and continual engulfment capacity. Macrophage-differentiation conditions, stimulation duration, culture density, and serum source should be standardized in experiments.

8 Disease Research and Targeting Potential

8.1 Autoimmune Diseases

Insufficient efferocytosis can cause accumulation of apoptotic cells and cellular debris, increase exposure of nucleic acids and other autoantigens, and allow damage-associated molecules released during secondary necrosis to activate innate immunity and promote autoantibody production. Defective dying-cell clearance in diseases such as systemic lupus erythematosus may simultaneously involve increased dying-cell generation, abnormal phagocytic receptors, insufficient lysosomal processing, and disruption of immune tolerance.

8.2 Atherosclerosis

Atherosclerotic plaques contain large numbers of apoptotic macrophages, foam cells, and smooth muscle cells. Effective efferocytosis during early disease stages can limit accumulation of cellular debris, whereas defective efferocytosis in advanced plaques can promote secondary necrosis and necrotic-core formation. Abnormal MerTK function, phagocyte lipid overload, and insufficient ABCA1-mediated cholesterol efflux can all aggravate plaque inflammation and instability.

8.3 Neurodegenerative Diseases and Retinal Degeneration

Microglia and other nervous-system phagocytes need to continuously clear dying neurons, synaptic structures, and myelin debris. Insufficient efferocytosis may cause accumulation of cellular debris and inflammatory mediators and interfere with neural-tissue repair. Retinal pigment epithelial cells and retinal phagocytes also perform continual clearance. Abnormal phagosome maturation or lysosomal degradation can cause accumulation of undegraded material and promote retinal degeneration.

8.4 Tumor Immune Microenvironment

Tumor tissues contain large numbers of cells that die because of hypoxia, nutrient deprivation, or treatment. Macrophage clearance of these cells can reduce secondary necrosis but may also promote anti-inflammatory signals such as IL-10 and establish an immunosuppressive microenvironment. Tumor cells can also inhibit macrophage phagocytosis through high CD47 expression. Therefore, the CD47-SIRPα axis, TAM receptors, and LAP-associated pathways are important research directions in tumor immunity.

8.5 Targeted Intervention Strategies

In autoimmune diseases, chronic inflammation, and atherosclerosis, strategies that enhance PS recognition, improve MerTK signaling, promote phagosomal degradation, or restore phagocyte metabolic adaptation can be investigated. In tumors, it may instead be necessary to block CD47-SIRPα, regulate TAM-receptor signaling, or alter immunosuppressive dying-cell processing. The direction of intervention may differ among diseases and should be designed according to disease stage, phagocyte type, and immunological endpoints.

9 Products

9.2 Products for Efferocytosis Receptors and Recognition Signals

9.3 Products for Rac1 and Actin-Cytoskeleton Research

9.4 Products for Phagosome, Lysosome, and LC3-Associated Research

9.5 Products for Inflammatory and Immunometabolic Detection

Efferocytosis research should simultaneously confirm the status of apoptotic target cells, phagocyte recruitment, and dying-cell recognition; distinguish surface binding from true internalization; and further validate phagosome acidification, lysosomal fusion, and dying-cell degradation. Only by integrating receptor dependence, actin-cytoskeleton remodeling, LAP-associated mechanisms, and immunometabolic outputs into the same evidence chain can the functions of efferocytosis in tissue homeostasis, inflammation resolution, and disease progression be accurately evaluated.

References

[1] Mehrotra P, Ravichandran KS. Drugging the efferocytosis process: concepts and opportunities. Nat Rev Drug Discov. 2022;21(8):601-620.

[2] Boada-Romero E, Martinez J, Heckmann BL, Green DR. Mechanisms and physiology of the clearance of dead cells by efferocytosis. Nat Rev Mol Cell Biol. 2020;21(7):398-414.

[3] Evans AL, Heit B. Quantitative Efferocytosis Assays. Methods Mol Biol. 2017;1519:25-41.

[4] Raymond MH, Ravichandran KS, et al. Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo. Science. 2022;375(6585):1182-1187.

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