When the process of apoptosis occurs, the ACs will generate and release some chemokines or other signals to recruit phagocytic cells. Meanwhile, in mammals, in addition to releasing find me to attract certain phagocytic cells, ACs also release “keep out” signaling factors to block the approach of inflammatory cells such as neutrophils. Lactoferrin, a multifunctional glycoprotein, is the only protein identified to date that acts as a “keep out” signal. “Find me” signalincludes lysophosphatidylcholine (LPC) (Lauber et al., 2003) and sphingosine 1-phosphate (S1P), nucleotides (including ATP and UTP 16), and chemokine CX3CL1 (fractalkine), etc. It has been shown that H₂O₂ may act as a “find me” signal in Drosophila embryos, which is required for the recruitment of blood cells to the wound area (Moreira et al., 2010). During the H₂O₂-induced wounding response, Src42A-Draper-Shark signaling was found to be essential for the hemocyte recruitment (Evans et al., 2015).

The cellular environment is very complex, including healthy cells, ACs, lymphocytes, and phagocytic cells. Therefore, the recognition of ACs by phagocytes is very crucial, and this process mainly depends on the molecules exposed to the apoptotic cell surface such as phosphatidylserine, Intercellular adhesionmolecule 3 (ICAM3) (Kristóf et al., 2013), Calreticulin (Gardai et al., 2005), oxidized low-density lipoprotein (Di and Maiseyeu, 2021), glycosylated surface proteins, etc. These signal molecules can be usually perceived as “eat me” signals, which distinguish ACs from healthy cells are easily recognized by phagocytes.

The most well-known and highly conserved “eat me” signal is phosphatidylserine (PS), phagocytes directly or indirectly recognize and bind these signals through their own PS recognizing membrane receptor (PSR), thereby promoting the recognition and engulfment during apoptosis and preventing inflammation. The trigger receptor and advanced glycation end product receptor of Brain Angiogenesis Inhibitor 1/3(BAI1/3), T cell immunoglobulin mucin receptor 4 (TIM4), Stabilin-1/2 (Stab2), (TREM)-like protein 2 (TLT2) are identified as directly binding to PS (Miyanishi et al., 2007; Park et al., 2007; Park et al., 2008; He et al., 2011). The recognition between PS and phagocytic receptors can also be mediated by bridging molecules, such as phagocytic receptor α _v β₃ integrin binds to ACs through PS-dependent bridging molecule MFG-E8; TAM receptor (receptor tyrosine kinase Mer Tyro3 and Axl, known as TAM receptors) recognizes PS through interacting with growth arrest-specific gene 6 (Gas6) or protein S (Savill et al., 1990; Nakano et al., 1997; Scott et al., 2001; Hanayama et al., 2002). Other bridging molecules including C1q, MBL, TSP-1 and TTR52 bind to phagocytic receptors LRP1, CRT, CD36 and MEGF10, respectively, in order to recognize PS (Savill et al., 1992; Ogden et al., 2001; Fond and Ravichandran, 2016). These receptors facilitate individually or coordinate with bridging molecules to promote the recognition of ACs.

We found that receptors on the surface of phagocytes are also highly conserved, such as in Drosophila Draper (Drpr, MEGF10 in mammals, CED-1 in worms), integrin and Croquemort (Crq, CD36 in mammals) and other phagocytosis receptors, which were found to function in different ways during phagocytosis: BAI1 is essential for phagosome formation, while TIM-4 stabilizes phagosomes (Mazaheri et al., 2014). In addition, healthy cells have a “Don’t eat me” signal that acts as an inhibitor to prevent phagocytosis by phagocytes, such as CD31, CD46 and CD47 (Poon et al., 2014).

Then macrophages integrate various signals from ACs and transmit them to the downstream, promoting a series of processes to recognize and engulf ACs.

Macrophages integrate signals from ACs to promote cytoskeletal rearrangement, a process that has been studied in the model organisms C. elegans and Drosophila, as well as in mammals. In C. elegans, upstream signals were found to converge in two parallel and independent signaling pathways:CED-2, CED-5, CED-12 pathway and CED-1, CED-6, CED-7 pathway which both subsequently activate CED-10, an evolutionarily highly conserved GTPase (Park and Kim, 2017) and thus stimulates skeletal rearrangement to form phagocytic vesicles (Wu and Horvitz, 1998a; Wu and Horvitz, 1998b; Liu and Hengartner, 1998; Reddien and Horvitz, 2000; Gumienny et al., 2001). CED-2/CED-5/CED-12 homologous signaling pathways in Drosophila and mouse were CG1587/myoblast city/Dmel, RKII/Dock180/ELMO1, and CED-1/CED-6 homologous signaling pathways were Drpr/dCed-6, MEGF10/GULP1 (Zheng et al., 2017), which are are highly conserved to regulate ACs clearance. Interaction between ABL-1 and ABI-1 inhibits ABI-1 and thus negatively regulates phagocytosis, but the study of this pathway in Drosophila and mammals remains to be determined (Hurwitz et al., 2009).

The dynamics of PtdIns (4,5) P2 and PtdIns3P phosphatidylinositol is a very important event in the sealing of phagocytic vesicles (Flannagan et al., 2012; Cheng et al., 2015; Wang and Yang, 2016). PtdIns (4,5) P2, PtdIns3P are abundantly present in unconfined and confined phagosomes respectively. The model organism C. elegans has been relatively well studied in this regard. With the accumulation of PtdIns3P, the phagosome recruits SNX9 family protein LST-4 to the phagosome, and SNX9 further recruits DYN-1 to complete phagocytosis. Researches in C. elegans, Drosophila and mammalian cells show that abnormal Dyn-1 function results in stalled phagosome maturation and aggregation of apoptotic bodies within phagosomes, indicating that it plays a key role in phagosome maturation (Kinchen et al., 2008; Yu et al., 2008; Shklover et al., 2015).

The maturation of phagosomes undergo several processes, including early endosome, late phagosome and phagolysosome formation (Almendinger et al., 2011; Cheng et al., 2015). The different forms of membranous vesicles require a series of Rab GTPases proteins, which also participate in the acidification of phagocytic lysosomes. For example, the GTPases RAB-5 and RAB-7 bind to early and late endosomes respectively to mediate the processes (Li et al., 2009). It was shown that early phagosome recruits RAB-5 protein to assemble downstream factors while RAB-7 participates in the later stages of phagosome maturation and mediates phagocytosis and lysosomal fusion (Vieira et al., 2002; Kinchen and Ravichandran, 2008). In mammalian the Mon1 interacts with Rab5, and the Mon1-Ccz1 complex binds Rab7 and may affect Rab7 activation, so Mon1-Ccz1 may facilitate the transition from Rab5 to Rab7 through a Rab exchange mechanism, but whether SAND-1-CCZ-1 (Mon1-Ccz1) uses a similar mechanism to regulate phagosome formation in S. hidradiata remains to be determined (Kinchen and Ravichandran, 2010; Zheng et al., 2021).

Not only RAB-5, RAB-7 and LAMP-1 are recruited during the process of phagosome maturation, but also V-ATPase and other factors such as histone proteases. In nematode and zebrafish studies, V-ATPase was revealed to play a role in different stages of phagosome formation. In zebrafish V-ATPase is not only involved in the acidification of lysosomes, but may also play a role in phagosome maturation. In nematodes, it is required in the early stages of phagosome maturation (Zheng et al., 2021). After Rab7 recruitment to the phagosome, the HOPS complex begins to recruit, thereby activating Rab7 and facilitating its eventual fusion with the lysosomal structure (Kinchen and Ravichandran, 2008).

After phagocytic lysosome formation, the phagocytic lysosome recruits proteins to the surface and releases acid hydrolases, among others, to degrade ACs. In C. elegans, LAAT-1, the lysosomal lysine/arginine transporter maintains lysosomal persistence and releases lysosomal histone protease L (CPL-1) and DNase II(NUC-1) to control the digestion and degradation of ACs (Liu et al., 2012; Xu et al., 2014).

Finally, when apoptotic cells are degraded within the phagolysosomes, a large amount of metabolic cargo is produced, such as amino acids, lipids, nucleic acids and some other potentially cytotoxic macromolecules, so macrophages induce ABCA1 expression, allowing cholesterol efflux and reducing damage to membranes by harmful substances (Kiss et al., 2006; Han and Ravichandran, 2011). During the clearance of apoptotic cells, the vesicular cycle in the cell is also not negligible, and the process is regulated by the RAB family of GTPases. RAB17 is recruited into the phagosome containing apoptotic cells, thus mediating a vesicular cycle from the phagosome to the recycling endosome for the purpose of returning the cell surface area (Yin et al., 2019).

During efferocytosis, ACs can attract phagocytes by releasing “find me” signals, such as nucleotides, chemokines and their modified membranes. These molecules can stimulate the migration of macrophages to ACs, but the recruitment of find me signals to macrophages depends on many other factors, such as the type of phagocytes and ACs and the stimulation of apoptosis (Ravichandran, 2010).

S1P, a type of lysophospholipid, is a sphingosine metabolite produced by sphingosine kinase (SphK, SphK1/SphK2) acting on sphingosine as an important “find me” signal to recruit phagocytes (Gude et al., 2008; Luo et al., 2016). S1PR, the receptor for S1P, which belongs to the G protein-coupled receptor family, is required for cell survival, cell migration, apoptosis, and inflammation through binding to S1P. During apoptosis induced by DNA damage, p53 accumulates which in turn activates the lysosomal pathway as well as the mitochondrial pathway. The mitochondrial pathway causes caspase enzyme activation and the lysosomal pathway releases cathepsins into the cytoplasm. The proteases released by both pathways cause downregulation of SphK1, which in turn reduces intracellular S1P levels (Taha et al., 2004). However, it is not clear whether the regulation of SphK1 by proteases occurs through direct cleavage or some other indirect mechanism. Binding of extracellular S1P to S1PR reduces the production of pro-inflammatory factors and anti-inflammatory factors (e.g., IL-10), vascular endothelial growth factor (VEGF), nuclear transcription factor peroxisome proliferator-activated receptor λ (PPARλ), and erythropoietin EPO is also upregulated, thereby stimulating the anti-inflammatory macrophage phenotype, promoting the polarization of macrophage M2, and enhancing efferocytosis (Cheng et al., 2015). S1P also inhibits macrophage death, induces COX-2 expression, promotes cAMP production, and inhibites NF-κB signaling. In addition, binding of extracellular S1P to S1PR1 promoted phagocytic vesicle maturation after pathogen uptake (Weigert et al., 2009).

Nucleotides can also be used as find me signals to recruit phagocytes (Elliott et al., 2009). Nucleotides are released extracellularly in a time- and caspase-dependent manner at low levels of ATP as well as UTP. In ACs, the plasma membrane channel Pannexin-1 (PANX1) mediates the release of ATP and UTP by forming hexameric channels in a caspase-dependent manner, promoting the releasing of nucleotides, thus mediates the recruitment of macrophages by binding to the nucleotide receptor P2Y2 (Chekeni et al., 2010). Previous study showed the expression of Panx1 was initiated and activated by transcription factors CREB and ETV4 in the rat epididymis (Dufresne and Cyr, 2014), which may give us an inspiration on the regulatory pattern of find me signals. The conversion of extracellular ATP into adenosine, which is the result of the interaction between CD39 and CD73 (Idzko et al., 2014). Adenosine binds to the adenosine A2A receptor on the surface of macrophages and subsequently inhibits NF-κB signaling and upregulates the expression of Thbs1 and the nuclear receptor gene Nr4a. Thbs1 is a major activator of TGFβ, while members of the Nr4a family restrain the level of pro-inflammatory cytokines such as TNFα and IL-8 in macrophages and promote the efferocytosis process (Elliott et al., 1950; Yamaguchi et al., 2014) (Figure 2).

Since the research on find me signaling is relatively limited, the specific release mechanism of “find me” signaling during recruitment and how transcription factors regulate the process of recruiting macrophages remain to be discovered, which means that this area is well worth investigating.

The migration and proximity of phagocytes to ACs depends on find me signals, while the specific recognition and binding to phagocytes depends on the exposure of eat me signals. The most studied and well-known eat me signal- PS, exposed on the cell surface of ACs by the co-regulation of phospholipid scramblase and flippase depending on the caspases activity (Segawa and Nagata, 2015).

Phagocytes recognize and bind these signals via their own PS recognition membrane receptors directly or indirectly via bridging molecules, thereby promote the clearance of ACs and prevent inflammation. Over the past few years, scientists realized that various receptors on phagocytes can recognize PS exposed on ACs. Tim4 expressed in lymphocytes of various mouse tissues, was found to bound and engulf ACs through recognizing PS by its immunoglobulin domain (Miyanishi et al., 2007). BAI1, which belongs to the adhesion-type G-protein-coupled receptor family, was reported as a PS recognition receptor, which formed a trimeric complex with ELMO and Dock180 to facilitate engulfment of ACs (Park et al., 2007). Stab2 which expresses in human monocyte-derived macrophages, mediates the clerance of aged red blood cells and ACs by recognizing PS (Park et al., 2008). MFG-E8 can simultaneously recruit integrin α_vβ₃ on phagocytes and recognize PS to uptake thre ACs. Gas6 and protein S, are involved in concatenate PS exposed on ACs to TAM receptors on phagocytes as bridging molecules (Anderson et al., 2003; Hanayama et al., 2004; Toda et al., 2012).

The expression of these receptors, and bridging molecules, enhances phagocyte recognition of ACs. Transcription factors such as the nuclear receptor super-family, STAT family and NF-κB have been found to be involved in the regulation of PSR, complement molecules and other eat me signals, which are important for the regulation of inflammation, the efficient and timely elimination of ACs and the stability of body’s immune system.

The peroxisome proliferator-activated receptor (PPAR) and the liver x receptor (LXR), which belong to the nuclear receptor superfamily, are involved in cellular lipid homeostasis. PPAR (heterodimers α, β/δ and γ) and LXR (heterodimers α, β) are ligand-activated transcriptional activators with different tissue expression. After binding to ligands such as fatty acids and oxysterols, PPAR and LXR then form heterodimers with the retinoid X receptor (RXR) and recruit co-activators to induce transcription of various genes involved in lipid and cholesterol metabolism (Han and Ravichandran, 2011). During ACs clearance, LXR and PPAR activate form heterodimers with RXR and upregulate phagocytic receptors and regulators. Existing studies have shown that activation of LXR and PPAR during apoptotic cell clearance leads to upregulation of phagocytic receptors (e.g.,: Mer) as well as of modulators (C1qb,Gas6,MFG-E8d) (A-Gonzalez et al., 2009; Mukundan et al., 2009). Here the clearance of apoptotic cells is influenced by two main pathways. In the first pathway, phagocytosis of apoptotic cells leads to the activation of LXR as well as PPAR, which upon activation regulates the expression of relevant phagocytic receptors as well as regulators. In the second pathway, recognition and binding of phagocytic receptors to phosphatidylserine activates LXR and PPAR, which in turn promotes the expression of Abca1, which not only induces cholesterol efflux but also promotes efferocytosis (Hamon et al., 2002). However, it is still unclear what ligands LXR and PPAR bind, how LXR and PPAR receive signals delivered by phosphatidylserine, and how they are transformed into macrophage lipid metabolism. Deficiency of RXR affects the transcription of cell surface receptors (e.g., CD36, Fcgr1, MERTK, Axl, etc.), regulators (e.g., C1qa, C1qb, C1qc, etc.) and transglutaminase-2 (Tgm2), which play important roles in cytokinesis and other macrophage functions.

PPAR-δ, PPAR-γ and RXRα have been demonstrated to upregulate MERTK and AXL transcriptional levels in macrophages (Han and Ravichandran, 2011). Macrophages lacking PPARγ, PPARβ/δ or RXRα (Vaquerizas et al., 2009; Gutiérrez-González et al., 2019), or PPARγ (Naeini et al., 2020) affect Axl transcription and inhibit ACs uptake (Röszer, 2017), causing macrophage adhesion and migration to be blocked (Garabuczi et al., 2015; Röszer, 2017). During efferocytosis, Axl and Mer signaling pathways directly inhibit Toll-like receptor (TLR) and type I IFN-driven inflammatory signaling pathways by different mechanisms. In dendritic cells, activation of Mer by ACs inhibits the IκB kinase IKK activity downstream of TLR4, suppressing NF-κB and reducing its binding to the TNF promoter. In addition, the reduction of TNFα is mediated by activation of Axl receptor tyrosine kinase and inducing of Twist transcriptional repressor, which binds to the E box region of the TNF promoter and inhibits NF-κB-dependent transcription (Sen et al., 2007).

MERTK is indirectly controlled by the glucocorticoid GC, which has been shown to upregulate LXR/RXR expression and eventually increase the uptake of ACs. Glucocorticoids increase the phagocytic ability to ACs both in short-term and subsequent phagocytosis. Short-term phagocytosis is mainly elevated by increasing of MERTK and C1q expression levels, whereas sustained phagocytosis acts by promoting the expression of LXR, PPARδ and UCP2 (Garabuczi et al., 2015) (Figure 3).

Macrophages internalize pathogens or ACs through enveloping them into vesicles called phagosomes, which then fuse with lysosomes to mature into phagolysosomes and ultimately degrade the pathogens or ACs. The cellular mechanism has been revealed relatively clear and detailed, yet the roles of transcription factors in controlling apoptotic cell clearance are less reported (Figure 4). The most studied transcription factors, which regulate apoptotic cell clearance are PPARs, LXR, RAR, RXR and GR.

Researchers have found that the activation of Fcγ-receptor which mediated phagocytosis and endocytosis, causes nuclear transposition of TFEB, which enhances the expression of lysosome genes, TFEB silencing reduces the enhancements in degradation and bacterial killing mediated by Fcγ-receptor (Gray et al., 2016).

The expression of the class B scavenger receptor CD36 was regulated by transcription factors PPARγ and RXRα, as the Cd36 promoter includes a interaction element for PPARγ/RXRα heterodimers, thus activates the process of pathogen elimination and apoptotic cell recognition (Silverstein and Febbraio, 2009; Röszer, 2017). And, the homolog of CD36 in Drosophila, crq was found to be transcriptional regulated by GATA factor and its co-factor, Bfc, which interacts with Srp zinc finger domain to strengthen this binding; thus, they function together in boosting crq expression and efferocytosis (Zheng et al., 2021). In Drosophila, it has also been shown that Srp may regulate the expression of factors mediating phagosome maturation and apoptotic cell degradation, thus the process of ACs clearance may be influenced by its deletion (Shlyakhover et al., 2018).

Another phagocytotic receptor in Drosophia, Drpr, has been reported to be regulated by transcription factor Stat92E, which can directly binds to the promoter of drpr, and mediates glial phagocytosis to axonal debris (Doherty et al., 2014).

In studies of TIM-1-mediated acute kidney injury, binding of TIM-1 to ACs triggers TIM-1 phosphorylation and the recruitment of p85, which interact with each other to block TLR4 expression, or the phosphorylation and activation of NF-κB, resulting in an anti-inflammatory phenotype and phagocytosis (Yang et al., 2015).

Immunoglobulins G and M (IgG, IgM) and complement binding to ACs provide eat me signals for macrophages. Increased PPARγ and RXR ligands promote IgG or IgM recognition by ACs, while the absence of PPARγ or RXRα reduces the expression of complement factors (e.g., C1q), thus inhibits the binding and uptake of ACs by macrophages (Roszer et al., 1950; Mukundan et al., 2009).