The smell phase is the first step of phagocytosis. During this phase, dead or stressed cells secrete soluble mediators, termed “find-me” signals (Figure 1A), such as chemokines fractalkine CX3CL1 (Truman et al., 2008), lysophosphatidylcholine (Lauber et al., 2003; Gude et al., 2008), sphingosine-1-phosphate, and complement components (Surugiu et al., 2019) and the nucleotides ATP and UTP (Elliott et al., 2009; Chekeni et al., 2010). On the one hand, the “find-me” signaling mediators chemoattract phagocytes to migrate toward dead cells. On the other hand, these mediators induce cytoskeletal changes and promote the expression of phagocytic receptors and the digestion machinery in phagocytes, which prepares the phagocytes for subsequent engulfment and digestion (Medina and Ravichandran, 2016; Medina et al., 2020). In stroke models, dead/stressed neurons release the find-me signal lysophosphatidylcholine (LPC) by enhancing secretory phospholipase A2 group X. LPC chemoattracts microglia via G protein-coupled receptor 132 (GPCR132) or P2X purinoreceptor 7 (P2X₇) (Inose et al., 2015) (Figure 1A). Dead/stressed neurons also secrete CX3CL1, which chemoattracts microglia via the fractalkine receptor (CX3CR1) (Harrison et al., 1998; Soriano et al., 2002). Notably, CX3CL1 knockout mice display less infarct damage following 22 h of reperfusion after transient cerebral ischemia (Soriano et al., 2002). Sphingosine-1-phosphate (S1P) likely acts as a finding-me signal via S1P receptor 2 (S1P₂) since the blockade of S1P₂ attenuates microglial recruitment and activation following stroke (Sapkota et al., 2019) (Figure 1A). The complement component C3a released from dying cells also chemoattracts microglia following stroke, while inhibition of the C3a receptor (CR3) with an antagonist for 2 days suppresses microglial recruitment and subsequent phagocytosis in a mouse cerebral ischemia model (Surugiu et al., 2019) (Figure 1A). In addition, knockout of CR3 reduces white matter injury by dampening microglia-mediated excessive phagocytosis of intact myelin at day 28 after ischemia in a mouse model (Zhang et al., 2020a).

Dying or stressed neurons also secrete nucleotides such as ATP and ADP, which chemoattract microglia via the P2Y₁₂ receptor (Webster et al., 2013) (Figure 1A). Consistently, knockout or inhibition of the P2Y₁₂ receptor suppresses microglial migration and clustering around damaged neurons, consequently decreasing damage at 3 days following transient cerebral ischemia (Webster et al., 2013). Find-me signals contribute to the anti-inflammatory properties of phagocytes (Medina and Ravichandran, 2016; Medina et al., 2020). It should be emphasized that nucleotides released in small amounts from cells during the very early stage of death (when cells are still intact) act as “find-me” signals, which are distinguishable from ATP released at high extracellular concentrations after complete cell lysis (Mehrotra and Ravichandran, 2022). Extracellular ATP at high concentrations is well known to be proinflammatory. However, recent research has suggested that pannexin-1-mediated release of ATP from viable cells is critical for suppressing airway inflammation (Medina et al., 2021). Notably, an inhibitor of pannexin-1 inhibits ATP release, suppresses neuroinflammation and improves outcome at 3–5 days postinjury in a mouse model of traumatic brain injury, suggesting that pannexin-1-mediated release of ATP contributes to neuroinflammation after brain injury (Garg et al., 2018). Thus, how pannexin-mediated ATP modulates phagocytosis and impacts neuroinflammation after stroke remains to be investigated.

The exposure of “eat-me” signals on the cell surface determines whether dead cells can be recognized by phagocytes. The “eat-me” signals are recognized by a cohort of phagocytic receptors. The most potent “eat-me” signal phosphatidylserine (PS) is evolutionarily conserved and present on the outer leaflet of the plasma membrane of apoptotic cells (Fadok et al., 1992). Conversely, PS is on the inner leaflet of the plasma membrane in live cells. PS asymmetry is actively maintained via an energy-dependent mechanism by specific enzymes such as flippases and scramblases (Suzuki et al., 2013b; Suzuki et al., 2014; Segawa and Nagata, 2015; Arashiki et al., 2016). PS exposure causes phagocytosis of dead cells either directly through PS receptors or indirectly through adaptor proteins (opsonins) (Figure 1B). In dying cells, caspase 3 induces the cleavage and inactivation of flippases and simultaneously activates scramblases (Suzuki et al., 2013a). TMEM16F is a calcium-activated phospholipid scramblase. The level of TMEM16F protein is increased, and reversible PS exposure is confirmed in neurons following ischemic stroke. Inhibition of PS exposure by knocking down TMEM16F in neurons dampens phagocytosis, rescues stressed but viable neurons in the penumbra 3 days after ischemia, and consequently decreases infarct damage and improves functional recovery (Zhang et al., 2020b). Notably, PS exposure also occurs in erythrocytes leaked into the parenchyma following intracerebral hemorrhage (ICH). Clearance of erythrocytes by microglia/macrophages via PS-dependent mechanisms promotes hematoma resolution and facilitates functional recovery following ICH (Chang et al., 2018; Yan et al., 2022a). Notably, elevated calcium or oxidants or a decrease in ATP also drives reversible PS exposure on the cell surface of stressed but still viable neurons following sublethal cellular stimuli (Neher et al., 2011). This induces phagocytes to engulf stressed but viable neurons, consequently leading to delayed neuronal death, brain atrophy and behavioral deficits at 28 days after stroke (Neher et al., 2012; Neher et al., 2013). For instance, subtoxic levels of glutamate induce neurons to reversibly expose PS on their surface and subsequently induce phagocytosis of stressed but viable neurons by microglia. Consequently, this kind of phagocytosis can be attenuated by blocking PS exposure (Neher et al., 2013).

Although PS is the major “eat-me” signal, a cohort of other “eat-me” signals also contribute to the recognition of dead cells by phagocytes (Mehrotra and Ravichandran, 2022). Other “eat-me” signals include calreticulin (Lauber et al., 2004; Arandjelovic and Ravichandran, 2015). Calreticulin, as an alternative “eat-me” signal, engages low-density lipoprotein receptor-related protein 1 (LRP1) on the cell surface of microglia/macrophages to initiate phagocytosis (Gardai et al., 2005) (Figure 1B). Calreticulin normally resides in the endoplasmic reticulum in healthy cells. It translocates to the cell surface upon endoplasmic reticulum stress, apoptosis, or proinflammatory signaling (Cockram et al., 2021). The calreticulin/LRP phagocytic signaling pathway mediates microglial phagocytosis of lipopolysaccharide- or Aβ-stressed neurons (Fricker et al., 2012). More relevantly, the signaling pathway likely plays a role in heme scavenging following ICH (Wang et al., 2018). The role of calreticulin in poststroke phagocytosis deserves further investigation. Notably, not all dead cells expose the same set of “eat-me” signals. Cell-exposed PS and a subset of other “eat-me” signals can trigger recognition by specific receptors on phagocytes (Mehrotra and Ravichandran, 2022).

PS exposed on the cell surface can be recognized by the receptors of phagocytes that bind PS directly, such as phosphatidylserine receptor (PSR), the T-cell immunoglobulin and mucin (TIM) family members, Stabilin2, and brain angiogenesis inhibitor I (BAI-I) (Fadok et al., 2000; Kobayashi et al., 2007; Miyanishi et al., 2007; Park et al., 2007; Park et al., 2008) (Figure 1B). PS can also be recognized via adaptor proteins such as milk fat globule-epidermal growth factor 8 (MFG-E8) and growth arrest-specific gene 6 (Gas6), which link to integrin or tyrosine protein kinase Mer (MerTK) receptors, respectively, on the phagocyte surface (Hanayama et al., 2002; Leonardi-Essmann et al., 2005; Brown and Neher, 2014) (Figure 1B). Notably, different cells express different sets of phagocytic receptors, which may depend on the stages of phagocytosis and tissue context. The binding of dead cells to phagocytic receptors triggers a complex array of cytoskeletal rearrangements, facilitating cargo engulfment. The signaling mechanism, the proteins involved and the kinetics of the process are highly variable following stroke and deserve further investigation.

Macrophages/microglia can also recognize and bind to apoptotic cells via scavenger receptors (Figure 1B). CD36 is a class B scavenger receptor. CD36 expression is low in the normal brain and significantly enhanced following stroke, mainly in macrophages in the brain (Kim et al., 2012). CD36 displays high affinity for many ligands, including apoptotic cells, and is well known to mediate the phagocytosis of erythrocytes following ICH (Zhao et al., 2009). CD36 knockout delays hematoma absorption and enlarges hematoma volumes at 5 days following ICH (Fang et al., 2014). Upregulating CD36 expression in microglia/macrophages via activation of the transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) promotes erythrocyte clearance at 7 or 10 days following ICH (Zhao et al., 2015). Bexarotene, a selective retinoid X receptor (RXR) agonist, also promotes erythrocyte phagocytosis, possibly via a mechanism related to CD36 upregulation 7 days following ICH (Chang et al., 2020). In contrast to the beneficial role of CD36 in ICH models, genetic deletion studies have shown that CD36 aggravates acute brain injury at 3 days after cerebral ischemia (Cho et al., 2005). However, CD36 may play a differential role in the recovery phase following cerebral ischemia. A study suggested that CD36 in macrophages mediates phagocytosis during the recovery phase and likely plays a reparative role via the resolution of inflammation following ischemic stroke (Woo et al., 2016). In conclusion, the scavenger receptor CD36, as an essential phagocytic receptor, exerts complex effects following stroke.

Opsonins associate with dead cells and thereby make dead cells visible to specialized phagocyte receptors. MFG-E8 and Gas6 are opsonins that are normally extracellular proteins. MFG-E8 recognizes and binds with PS exposed on neurons and therefore drives the microglial phagocytosis of PS-exposed neurons via the vitronectin receptors α_vβ₃ or α_vβ₅ on phagocytes (Neniskyte and Brown, 2013) (Figure 1B). Alternatively, PS exposed on neurons can be recognized by Gas6, which then induces microglial phagocytosis of neurons via the phagocytic receptor MerTK (Fourgeaud et al., 2016) (Figure 1B). The expression of MFG-E8 and MerTK is transiently enhanced in microglia/macrophages at 3–7 days after stroke, and knockout of either protein prevents delayed neuronal loss and brain atrophy and improves long-term functional deficits at 28 days after focal cerebral ischemia (Neher et al., 2013). This suggests that neuronal PS exposure drives phagocytosis of stressed neurons via opsonins after stroke and that blocking signaling is beneficial following cerebral ischemia.

The role of complement proteins such as opsonins in poststroke phagocytosis has been demonstrated by a recent study, which showed that targeted complement inhibition in the ischemic area salvages stressed neurons and inhibits neuroinflammation after ischemic stroke (Alawieh et al., 2018). Complement components, including C1q and C3, can be produced by microglia. The complement component C1q, as an opsonin, binds to eat-me signals, such as surface PS and calreticuline, to strengthen phagocytic recognition and promote phagocytosis (Cockram et al., 2021). C1q may also interact with the phagocytic receptor complement receptor 3 on the phagocyte surface to induce phagocytosis (Linnartz et al., 2012) (Figure 1B). C1q, as an opsonin, has an important role in synapse pruning. C1q binds to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx, and the elimination of desialylated neurites is mediated by the complement receptor CR3 (Linnartz et al., 2012). Notably, reducing C1q levels by inhibiting the classical complement pathway attenuates brain damage at 2 days after hypoxia in a mouse model of hypoxic ischemic encephalopathy (Kumar et al., 2021). Moreover, the serum levels of C1q correlate with the severity of neurological deficits and infarct sizes in human patients with ischemic stroke (Wang D D et al., 2020). These results suggest that opsonin C1q has great impacts on stroke outcomes.

The conversion of C3 to C3a and C3b is essential for complement-opsonin activation (Kumar et al., 2021). C3a recruits and activates microglia, whereas C3b promotes microglial phagocytosis via the CR3 receptor (Yu et al., 2022) (Figure 1B). Complement C3 inhibition specifically at synapses inhibits synaptic engulfment by microglia and reduces synapse loss in a mouse model of multiple sclerosis (Werneburg et al., 2020). More relevantly, C3a peptide treatment reduced tissue loss at 22 days after hypoxia–ischemia and improved memory impairment at 41 days after neonatal hypoxia–ischemia in wild-type mice but not in CR3-deficient mice (Jarlestedt et al., 2013), suggesting that complement C3a (C3a) acts through the receptor of C3a to protect against ischemic injury.

A recent study reported that S1P released from microglia may also function as a novel opsonin that engages triggering receptor expressed on myeloid cells 2 (TREM2) on microglia to promote microglial phagocytosis (Figure 1B). The study showed that microglia released apolipoprotein E-loaded S1P, which bound to apoptotic neurons via low-density lipoprotein receptor related protein 1B (LRP1B). Then, S1P binds to TREM2 on microglia to promote microglial phagocytosis of apoptotic neurons and thereby protect against ischemic brain injury at 48 h after cerebral ischemia (Xue et al., 2022). These results suggest complex roles of S1P in poststroke phagocytosis and pathology.

The exposure of “do not eat-me” signals on viable cells suppresses phagocytosis (Segawa et al., 2011) Numerous “do not eat-me” molecules have been discovered. These “do not eat-me” signaling molecules bind “anti-phagocytic receptors” on phagocytes to inhibit phagocytosis of live cells by phagocytes. For instance, CD47 on live cells binds to signal-regulatory protein-α (SIRPα) on macrophages (Figure 1D). SIRPα/CD47 binding induces tyrosine phosphorylation of the cytoplasmic domain of SIRPα, leading to the recruitment and activation of the phosphatases SHP1/2. SHP1/2 activation inhibits phagocytosis by suppressing non-muscle myosin IIA (Tsai and Discher, 2008). Knockout of the CD47 gene confers protection at 24 and 72 h after ischemia in a murine model of transient focal cerebral ischemia (Jin et al., 2009). Consistently, SIRPα deletion also confers robust neuroprotection at 24 h following cerebral ischemia (Wang et al., 2012). Thus, current evidence suggests that inhibiting SIRPα/CD47 signaling is neuroprotective following focal cerebral ischemia.

CD47 also acts as a “do not eat me signal” on erythrocytes and normally inhibits erythrophagocytosis via SIRPα expressed on phagocytes (Ni et al., 2016). In nude mice injected with blood from CD47 knockout or wild-type mice, CD47 knockout blood had quicker hematoma absorption than wild-type blood at 3 days after ICH (Ni et al., 2016). A CD47 blocking antibody also accelerated hematoma clearance in both young and aged mice (Jing et al., 2019; Tao et al., 2020). CD47 levels within the hematoma decreased with time in a pig ICH model (Cao et al., 2016). CD47 is also involved in erythrocyte clearance in other models of intracranial hemorrhage. In a rat model of intraventricular hemorrhage (IVH), the CD47 blocking antibody accelerated absorption of the intraventricular clot and reduced early erythrolysis at day 3 after IVH (Ye et al., 2021). Collectively, emerging evidence suggests that the “do not eat me” signal of CD47 delays erythrophagocytosis following hemorrhagic stroke.

Healthy cells have sialic acid residues that are integrated into glycoproteins and glycolipids residing on the cell surface (Figure 1D). Sialylation of glycoproteins and glycolipids inhibits phagocytosis via sialic acid-binding immunoglobulin-like lectins (Siglec) on the phagocyte cell surface, whereas desialylated glycoproteins residing on the cell surface promote phagocytosis by amplifying “eat-me” signals (Yu et al., 2022). Siglec-E is expressed in the brain, particularly on the surface of microglia. Genetic deletion of Siglec-E exacerbated neuronal death induced by oxygen-glucose deprivation in mouse primary cortical cultures, a mixed culture containing both neurons and glial cells. Moreover, compared to wild-type animals, Siglec-E knockout mice display more severe neurological deficits and larger infarct sizes at day 3 after cerebral ischemia (Li et al., 2022). Therefore, further investigation is needed to explore whether Siglec-E contributes to stroke outcomes by serving as a “do not eat me signal.”

Recently, the phosphatidylserine receptor CD300a was identified to function as an anti-phaocytic receptor during poststroke phagocytosis (Figure 1D). CD300a is highly expressed on infiltrated myeloid cells, including monocytes. CD300a can act through the CD300b-DNAX-activation protein 12 signaling pathway to inhibit phagocytosis of apoptotic cells. Deletion of CD300a enhanced phagocytosis by myeloid cells infiltrating the brain and consequently reduced the release of damage-associated molecular patterns from dead cells, attenuating inflammation in the penumbral region. An anti-CD300a neutralizing antibody also reduced infarction at 24 h after ischemia and ameliorated neurological deficits during the acute phase of ischemic stroke (Nakahashi-Oda et al., 2021). Therefore, current evidence suggests that “do not eat me” signals play a complex role following stroke.

Peroxisome proliferator-activated receptor-γ (PPARγ) and nuclear factor erythroid 2-related factor (Nrf 2) are the first two transcription factors identified to be involved in the regulation of phagocytic receptor expression following stroke. PPARγ displays multiple functions under pathophysiological conditions (Heming et al., 2018). PPARγ activation is an important mechanism underlying monocyte differentiation into macrophages with efficient phagocytic capacities (Cai et al., 2018). Notably, it has been shown that PPARγ activation promotes phagocytosis via the mechanism of CD36 upregulation, which is beneficial in both hemorrhagic and ischemic stroke models. PPARγ activation enhances the expression of the scavenging receptor CD36 in microglia/macrophages, facilitates the phagocytic clearance of erythrocytes, and consequently improves functional recovery in rodent models of ICH (Zhao et al., 2007). In animal models of cerebral ischemia, PPARγ activation by rosiglitazone also increases CD36 expression in microglia, which contributes to the resolution of inflammation and clearance of infiltrated neutrophils at 48 h following cerebral ischemia (Ballesteros et al., 2014). PPARγ activation likely increases CD36 expression via a 5-lipoxygenase (5-LO)-mediated mechanism. PPARγ activation induced 5-LO expression in neurons, and lipoxin A₄, one of the products of 5-LO, and lipoxin A₄ (Lipo A₄) enhanced CD36 independently of PPARγ (Ballesteros et al., 2014) (Figure 2A).

The transcription factor nuclear factor erythroid 2-related factor (Nrf2) plays a crucial role in antioxidant and anti-inflammatory responses. The Nrf2 agonist sulforaphane enhances phagocytosis of erythrocytes by upregulating the expression of CD36 in microglia after ICH (Zhao et al., 2015). Consequently, promoting hematoma clearance by Nrf2 activation improved outcomes following ICH. Moreover, CDDO-EA, a novel Nrf2 activator, also protects against ischemic injury by enhancing inducible heme oxygenase (HO-1) expression and microglial polar polarization toward the M2 phenotype at 48 h after ischemic stroke in mice (Lei et al., 2021). Thus, Nrf2 activation is an important regulatory mechanism underlying CD36-mediated phagocytosis following stroke.

Carbon monoxide (CO) and hydrogen sulfide (H₂S) are endogenous gasotransmitters displaying essential roles in multiple pathophysiological processes, along with the first identified gasotransmitter nitric oxide. Interestingly, both CO and H₂S have been shown to be involved in regulating phagocytosis following hemorrhagic stroke. Spontaneous hematoma resolution occurs both in human patients and animal models following cerebral hemorrhage. Theoretically, erythrophagocytosis plays an important role in spontaneous hematoma resolution. Nevertheless, the mechanisms underlying spontaneous hematoma resolution are poorly understood.

Subarachnoid hemorrhage (SAH) is a severe form of stroke that carries a mortality rate of 50%. Following SAH, heme released from extraversated erythrocytes functions as a disease-associated molecular pattern. Free heme is metabolized by heme oxygenase (HO), leading to the production of CO. In a mouse model of SAH, the expression of inducible HO (HO-1) in microglia is enhanced following SAH, which consequently attenuates neuronal cell death and vasospasm, improves cognitive function and promotes clearance of cerebral blood burden (Schallner et al., 2015). Inhalation of CO after SAH reduced injury by enhancing erythrophagocytosis in mice with HO-1 deletion in microglia. Clinical data from human SAH patients further revealed that HO-1 activity was markedly increased in cerebrospinal fluid (CSF). Moreover, cisternal hematoma volume was negatively correlated with HO-1 activity in the CSF of these patients. In conclusion, microglial generation of CO by HO-1 is a regulatory mechanism underlying erythrophagocytosis following SAH (Schallner et al., 2015). Mechanistically, CO enhanced microglial phagocytosis of erythrocytes by activating AMP-activated protein kinase (AMPK) to upregulate the expression of the phagocytic receptor CD36 in microglia following SAH (Kaiser et al., 2020) (Figure 2B). Collectively, current evidence suggests that the generation of endogenous gasotransmitter CO is an important regulatory mechanism underlying erythrophagocytosis and spontaneous hematoma resolution following SAH.

H₂S plays important roles in pathogenesis of the nervous system (Hou et al., 2017; Zhao et al., 2017). Notably, exogenous and endogenous H₂S has been shown to protect against injuries from both ischemic and hemorrhagic stroke (Wang et al., 2014; Zhang et al., 2017; Jia et al., 2020; Yan et al., 2022b). Recently, it was reported that ICH enhanced the expression of the H₂S synthase cystathionine β-synthase (CBS) and CBS-derived H₂S in brain-resident phagocytic microglia (Yan et al., 2022a). Notably, endogenous H₂S derived from CBS in microglia promoted continual phagocytosis of erythrocytes by microglia in vitro and contributed to spontaneous hematoma resolution at 5 days and 14 days following ICH in a mouse model. Mechanistically, oxidation of CBS-derived endogenous H₂S by sulfide-quinone oxidoreductase (SQR) initiated reverse electron transfer at mitochondrial complex I, resulting in enhanced superoxide production. Complex I-derived superoxide subsequently activated uncoupling protein 2 (UCP2) to promote erythrocyte phagocytosis by microglia (Figure 2C). UCP2 has been reported to be an essential mediator of continual phagocytosis by reducing mitochondrial membrane potential (Park et al., 2011). Moreover, hyperhomocysteinemia, an established stroke risk factor, is found to impair ICH-enhanced CBS expression and delay spontaneous hematoma clearance, while administration of an H₂S donor facilitated hematoma resolution in mice with hyperhomocysteinemia. The study showed that enhancing the production of endogenous H₂S following ICH is an endogenous regulatory mechanism underlying erythrophagocytosis and spontaneous hematoma resolution following ICH. For the first time, the study suggested a mechanism that regulates continual phagocytosis following stroke.

As a member of the signal transducer and activator of transcription family, signal transducer and activator transcription 6 (STAT6) is principally activated by interleukin-13 (IL-13) and interleukin-4 (IL-4) (Quelle et al., 1995). STAT6 exerts multiple functions in lymphocytes and myeloid cells (Goenka and Kaplan, 2011). The expression of STAT6 is enhanced, and its related signaling cascades are activated following both ischemic and hemorrhagic strokes, which mechanistically promote microglial/macrophage phagocytosis and functional outcomes. In animal models of cerebral ischemia, the STAT6/arginase 1 (STAT6/Arg1) pathway contributes to phagocytosis of dead/dying cells by microglia and macrophages, and upregulating STAT6/Arg1 signaling reduces brain infarction and facilitates long-term functional recovery at 3 and 7 days following ischemic stroke in mice (Cai et al., 2019). STAT6 is also an essential mediator of erythrocyte phagocytosis after ICH, and the IL-4/STAT6 pathway enhances long-term recovery in models of ICH (Xu et al., 2020). Enhancing the expression of interleukin-1 receptor-like 1 (ST2) is likely the key downstream mechanism underlying hematoma clearance enhanced by IL-4/STAT6 following ICH (Figure 2D).

Phagocytosis includes the smell, eating and digestion phases. Most studies on poststroke phagocytosis focus on the smell and eating phase, especially on the “find-me” and “eat-me” signals and the corresponding receptors that enable the sense and recognition of dead/dying cells by phagocytes. How the digestion phase is regulated and how digestion impacts phagocytosis and stroke outcomes are extremely understudied. Indeed, the most severe challenge for phagocytes following engulfment of dead cells is how to rapidly and efficiently process the almost doubled intracellular mass to maintain metabolic homeostasis (Han and Ravichandran, 2011; Mehrotra and Ravichandran, 2022). Mounting evidence suggests that the digestion phase is critical to phagocytosis. We currently lack an understanding of how the digestion phase is regulated in poststroke phagocytosis, and a better understanding of how digestion is regulated and impacts outcomes following stroke has important implications for novel therapeutic strategies for stroke. As we discussed above, lysosomes theoretically play essential roles in the digestion phase. TFEB, as the key master regulator of lysosome biogenesis, may essentially contribute to digestion during poststroke phagocytosis. More research will be needed to explore how TFEB regulates poststroke phagocytosis and how TFEB impacts brain injury repair via phagocytic mechanisms following stroke. Rab7 is primarily associated with late endosome structure and possibly is the only lysosomal Rab protein identified thus far (Zhang et al., 2009). Notably, a Rab7 antagonist ameliorates brain atrophy and improves neurologic deficits following ischemic stroke (Qin et al., 2019). Therefore, future research is needed to investigate whether Rab7 plays a role in regulating digestion following stroke. Other mechanisms, such as mechanisms underlying phagolysosomal assembly (Doran et al., 2020), may also markedly modulate the digestion phase and thus have significant impacts on poststroke phagocytosis. For instance, NADPH oxidase 2-derived superoxides have been reported to be essential for conjugating the autophagy-related protein LC3-II to the phagosomal membrane. The process, in turn, promotes phagolysosomal assembly and acidification, thereby facilitating digestion of phagocytic cargos (Bagaitkar et al., 2018). Thus, future research is needed to investigate how these signaling pathways impact stroke outcomes by modulating phagocytic digestion.

Compared to the large number of dead cells generated under pathological conditions, the number of phagocytes is rather limited (Park et al., 2011; Wang et al., 2017). For instance, it has been reported that a relatively small number of microglia/macrophages surround large volumes of hematoma at 3 days following ICH (Yan et al., 2022a). Efficient phagocytosis critically depends on the capacity of a single phagocyte to continually take up dead cells (Park et al., 2011; Wang et al., 2017; Mehrotra and Ravichandran, 2022). Thus, continual phagocytosis has significant impacts on stroke outcomes. However, how continual phagocytosis is regulated following hemorrhagic and ischemic stroke is extremely understudied. There are several challenges faced by phagocytes undergoing continual phagocytosis. First, each round of phagocytosis internalizes a substantial amount of plasma membrane as the phagosome enters the cell. Therefore, rapid restoration of the cell membrane is a prerequisite for the subsequent round of phagocytosis (Doran et al., 2020). Several mechanisms have been implicated to be essential for membrane restoration following phagocytosis. In macrophages undergoing continual phagocytosis, mitochondrial fission enhanced by elevated levels of the mitochondrial fission protein dynamin-related protein 1 leads to the restoration of the cell membrane by facilitating membrane recycling via promotion of phagolysosome-to-plasma membrane vesicular transport (Wang et al., 2017). The RAB17-dependent mechanism is also engaged in vesicular transport from phagolysosomes to recycling endosomes, which may contribute to the restoration of the plasma membrane (Yin et al., 2019). However, whether these mechanisms are also essential for continual phagocytosis following stroke or whether other novel mechanisms are engaged in poststroke phagocytosis needs further investigation. Second, degradation of engulfing material tremendously enhances metabolic cargo in phagocytes, including amino acids, lipids and nucleic acids, which must be processed in a safe and rapid manner (Han and Ravichandran, 2011; Doran et al., 2020). Mitochondria are essential for metabolism of metabolic loads. However, degradation of dead cells only results in a transient increase in the mitochondrial membrane potential (MMP) in phagocytes (Park et al., 2011), suggesting that a mechanism is activated to reduce excessive MMP. Enhanced expression of the mitochondrial uncoupling protein UCP2 was found to be the mechanism underlying MMP modulation during phagocytosis (Park et al., 2011). Notably, MMP was persistently high in phagocytes with UCP2 knockdown, and consequently, continual phagocytosis was impaired in these phagocytes. Although the signaling mechanisms linking UCP2-mediated MMP modulation to continual phagocytosis remain to be determined, the findings suggest a critical role of UCP2 in modulating continual phagocytosis. Indeed, the mitochondrial uncoupler-induced decrease in MMP has been shown to be protective following ICH (Pan et al., 2020). More relevantly, a recent study uncovered a new mechanism through which UCP2 activity rather than UCP2 expression was enhanced to promote continual phagocytosis of extraverted erythrocytes following ICH (Yan et al., 2022a). ICH was found to enhance the expression of the H₂S synthase cystathionine β-synthase (CBS) and CBS-derived H₂S in brain-resident phagocytic microglia (Yan et al., 2022a). Then, oxidation of CBS-derived endogenous H₂S by sulfide-quinone oxidoreductase initiates reverse electron transfer at mitochondrial complex I, resulting in enhanced superoxide production. Complex I-derived superoxide subsequently activates uncoupling protein 2 (UCP2) to decrease MMP after erythrocytosis, which promotes continual phagocytosis and hematoma clearance following ICH. The study suggested a regulatory mechanism underlying continual phagocytosis following stroke. However, our current understanding of the mechanisms underlying continual phagocytosis is rather limited. Therefore, more studies are needed to explore the regulatory mechanisms underlying continual phagocytosis following stroke. A better understanding of the mechanisms will advance the development of novel therapeutic strategies for treating stroke (Figure 3).

Phagocytosis resolves inflammatory responses and activates prorepair processes under pathological conditions. The proresolving effects of phagocytosis are attributed not only to the clearance of dead cells and consequent prevention of the release of potentially cytotoxic contents from dead/dying cells (Mehrotra and Ravichandran, 2022). Emerging evidence suggests that phagocytosis itself induces the proresolving and prorepair phenotype switch of professional phagocytes, such as macrophages, under pathological conditions. When the phenotype switch induced by phagocytosis is impaired, tissue repair and functional recovery are compromised, leading to impaired resolution of inflammation and development of disease. For instance, phagocytic clearance of dying cells directly induces the transition of cardiac macrophages into a phenotype with enhanced expression of vascular endothelial growth factor (VEGF) C, and macrophage-derived VEGFC inhibits inflammation after myocardial infarction (Glinton et al., 2022) (Figure 3). Mechanistically, metabolic reprogramming is the mechanism involved in the phenotypic switch during phagocytosis. Phagocytosis is reported to enhance interleukin-10 (IL-10) expression in macrophages after myocardial infarction, which helps resolve inflammation and promote repair (Zhang et al., 2019) (Figure 3). The elevation of interleukin-10 (IL-10) is independent of glycolysis but is bolstered by fatty acids derived from apoptotic cells, which are metabolized through mitochondrial β-oxidation and the mitochondrial electron transport chain to increase the coenzyme NAD⁺. Loss of IL-10 due to the defect in mitochondrial complex III can be rescued by adding NAD⁺ precursors. NAD⁺, in turn, acted through the sirtuin signaling cascade to activate the IL-10 transcription factor PBX1. These results suggest that digestion of phagocytic cargos and subsequent metabolism of metabolic loads by phagocytes induces phenotypic switching of phagocytes via metabolic reprogramming. Phagocytosis-induced phenotypic polarization of monocyte-derived macrophages (MDMs) also contributes to functional recovery and tissue repair after ICH. By profiling the transcriptional profiles of monocyte-derived macrophages in the mouse brain following ICH, phenotypic changes in infiltrating monocyte-derived macrophages were confirmed to be essential for hematoma clearance and neurological recovery (Chang et al., 2018). Notably, phagocytosis of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice, loss of the phagocytic receptor tyrosine kinases AXL and MerTK not only reduced phagocytosis but also decreased alternative activation of macrophages after ICH, which resulted in delayed hematoma clearance and neurological recovery. Thus, phagocytosis of apoptotic erythrocytes is responsible for macrophage phenotype switching and neurological recovery following ICH. However, the molecular mechanisms underlying the phenotypic remodeling of phagocytes following stroke are not clear. Mitochondria are the key organelles for processing phagocytic loads, and current research suggests that metabolic remodeling induced by mitochondrial catabolism of metabolic loads derived from phagocytosed cargos is essential to the phenotypic polarization of phagocytes after cardiac ischemia (Zhang et al., 2019). Thus, future research is needed to explore whether the phenotypic switch of phagocytes following stroke is mediated by metabolic reprogramming and to identify key master regulators of phenotypic switching following stroke. In particular, further research is needed to explore which specific phagocyte phenotypes induced by phagocytosis confer protective and prorepair effects and how to manipulate phagocyte phenotypes to promote recovery following stroke. A better understanding of these mechanisms will advance the development of novel therapeutic strategies for treating stroke. In addition, we want to emphasize that the phagocytosis-induced phenotypic switch is related to the degradation of dead cells and subsequent mitochondrial oxidation of metabolic loads (Chang et al., 2018). Thus, the strategy of using non-degradable beads in phagocytosis assays is not suitable for the investigation of poststroke phagocytosis, especially for the investigation of phenotypic switching. We and others have used apoptotic erythrocytes in assays of microglial phagocytosis in vitro models of intracerebral hemorrhage (Chang et al., 2018; Yan et al., 2022a). We recommend that using ingestible dead cells is more appropriate to measure the phagocytic function of microglia than using inert latex beads.

There are other knowledge gaps in the understanding of poststroke phagocytosis. For instance, it has long been recognized that phagocytosis function deteriorates during aging. In particular, CD22, as a negative regulator of phagocytosis, is reported to be upregulated in aged microglia. CD22 mediates the antiphagocytic effect of α2–6-linked sialic acid, and CNS delivery of a CD22 function-blocking antibody promotes the clearance of myelin debris, amyloid-β oligomers, and α-synuclein fibrils in vivo by reprogramming microglia toward a homeostatic transcriptional state (Pluvinage et al., 2019). However, it remains to be investigated how aging-related deterioration of microglial phagocytosis function impacts stroke outcomes and what are the key mediators that play essential roles in aging-related deterioration of microglial phagocytosis following stroke.

In addition, autophagy, a major intracellular degradative pathway, is relatively similar to phagocytosis. Although the goal of autophagy is to degrade intracellular cargo rather than extracellular cargo, autophagy and phagocytosis share some mechanisms and cellular machinery. Interestingly, recent studies have reported that autophagy is directly relevant to phagocytosis after stroke. Basal autophagy was reported to be critical for maintaining microglial physiology, including phagocytosis. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by ischemic stroke in vivo (Beccari et al., 2023). However, the mechanisms by which autophagy modulates phagocytosis following stroke warrant further investigation.

Notably, both peripheral-infiltrating myeloid cells and brain-resident microglia are involved in debris clearance via phagocytosis following stroke, and some controversial results may stem from the overlapping functions of peripheral myeloid cells and microglia. Whether microglia or macrophage phagocytosis is dominant following stroke is controversial. For instance, compared to microglia, infiltrating monocyte-derived macrophage (MDM)-mediated phagocytosis is reported to play a predominant role in pathology and recovery following stroke (Park et al., 2022). Moreover, it has been shown that MDMs have higher phagocytic activity and likely play a more important role in erythrophagocytosis following ICH (Chang et al., 2021). However, it is also reported that brain-resident microglia are the dominant phagocytes responsible for erythrophagocytosis following ICH and SAH (Schallner et al., 2015; Yan et al., 2022a). However, most studies do not distinguish between microglial and macrophage phagocytosis following stroke. Thus, more research is needed to distinguish the phagocytic roles of microglia and macrophages and to explore whether the phagocytic functions of microglia/macrophages can be individually manipulated. In addition, it also remains unclear how professional phagocytes and non-professional phagocytes cooperate to perform phagocytic functions following stroke. These issues have been reviewed (Jia et al., 2021; Chen et al., 2022; Yu et al., 2022), and we did not discuss them in detail here.

In conclusion, a number of phagocytic signals orchestrate the regulation of phagocytosis following stroke, and poststroke phagocytosis exerts multifaceted effects on stroke outcomes (Table 1), which are context-dependent and perhaps cell type dependent. Consequently, it represents a challenge to balance these effects to establish a milieu that is favorable for brain repair and functional recovery. Although much has been learned about the molecular mechanisms governing the sense and recognition of dead cells by phagocytes following stroke, some key areas remain extremely understudied, including the mechanisms involved in digestion regulation, continual phagocytosis and phagocytosis-induced phenotypic switching following stroke. Understanding these knowledge gaps holds promise for uncovering new biological targets for stroke treatment.