The Role of Microglial Phagocytosis
in Ischemic Stroke
小胶质细胞吞噬功能在缺血性脑卒中中的作用
翻译：研究生：张洮铭；科研秘书：刘虹；导师：陈晨
导语：缺血性脑卒中是全球严重的长期残疾和死亡的主要原因，也是我院神经内科收治最多的病种，这篇来自1区7.3分的2022年最新文献提示，小胶质细胞的吞噬作用是免疫炎症和脑卒中恢复的双刃剑。小胶质细胞通过吞噬细胞碎片、清洁浸润的中性粒细胞和为神经发生创造最佳微环境来限制炎性损伤。因此，了解其分子机制并调节选择性小胶质细胞吞噬功能是一个极具吸引力的靶点，在卒中后具有较长的治疗窗口期。
高质量的文献和研究既提示我们学科融合、学科交叉的重要性，也给我们科学研究带来了新的思路。通读本文后，给我们研究者带来继续进行深入研究的思路和冲动。身处心血管病专科医院，上述病例和数据不难获得，要的就是十年如一日的科研坚持和随访。
神经内科3病区目前在研科研项目4项，其中卫健委“四个一批”重大科技攻关项目一项。今年出版专著1部，待出版专著2部，已发表SCI论文3篇，最高为2区6.05分的脑卒中代谢学相关研究。我们有完备的科研团队和研究生团队欢迎兄弟科室常来常往，交流科研经验，共同进行学科融合和多学科交叉科研思路的探讨起申请立项学科交叉的课题。
下面我们就来阅读整篇文献。
Microglia are the resident immune cells of the central nervous system that exert diverse roles in the pathogenesis of ischemic stroke. During the past decades, microglial polarization and chemotactic properties have been well-studied, whereas less attention has been paid to phagocytic phenotypes of microglia in stroke. Generally, whether phagocytosis mediated by microglia plays a beneficial or detrimental role in stroke remains controversial, which calls for further investigations. Most researchers are in favor of the former proposal currently since efficient clearance of tissue debris promotes tissue reconstruction and neuronal network reorganization in part.
Other scholars propose that excessively activated microglia engulf live or stressed neuronal cells, which results in neurological deficits and brain atrophy. Upon ischemia challenge, the microglia infiltrate injured brain tissue and engulf live/dead neurons, myelin debris, apoptotic cell debris, endothelial cells, and leukocytes. Cell phagocytosis is provoked by the exposure of “eat-me” signals or the loss of “don’t eat-me” signals. We supposed that microglial phagocytosis could be initiated by the specific “eat-me” signal and its corresponding receptor on the specific cell type under pathological circumstances. In this review, we will summarize phagocytic characterizations of microglia after stroke and the potential receptors responsible for this programmed biological progress.
Understanding these questions precisely may help to develop appropriate phagocytic regulatory molecules, which are promoting self-limiting inflammation without damaging functional cells.
Keywords: phagocytosis, microglia, ischemic stroke, signaling receptors, prognosis
Stroke has a great impact on public health and is still the leading cause ofdeath and disability in the world. Obstruction of the blood and oxygen supply after ischemic stroke is the initial reason for cell death. The intense changes of circumstances like accumulation of cell debris or infiltration of immune cells in the brain may induce the secondary progression of cell injury. Strategies aiming at restoring cerebral perfusion, namely, intravenous rt-PA injection and emergent endovascular intervention have been written in stroke therapeutic guidelines. Plenty of stroke patients cannot receive this treatment for missing the time windows although sizable efforts have been made to shorten the door to needle time (DNT). Hence, it is imperative to develop drugs that extend the time window through alleviating the secondary injury and promoting rehabilitation of ischemic stroke.
Microglia, the professional phagocyte ofthe brain, are capable of engulfing cell debris and pruning synapses in developing brain and various cerebral diseases. Despite being less commonly known, a growing body of literature has linked microglial phagocytosis with stroke recovery. In this review, we will summarize phagocytic characterizations of microglia after stroke and the potential receptors responsible for this programmed biological progress.
Originating from myeloid precursors, microglial cells born in the yolk sac invade the central nervous system (CNS) during early embryonic development, which continuously supervises the brain parenchyma and protects against damage-associated pathogens. Upon ischemic stroke, microglia are rapidly activated and accumulate in large numbers at the site of infarction, which is also defined as microgliosis. Whether microglia are replenished in situ or derived from circulatory precursors is the subject of great interest. Taking advantage of a mouse photothrombosis stroke model, Li et al. reported that reactive microglia increased within minutes and were recruited to the infarcted area continuously during the first week after stroke induction. Additionally, local resident microglia division rather than recruitment of circulating macrophage was the main source of microgliosis. Researchers then begin to search whether microglia progenitors exist in the brain, which
gives rise to the newborn microglia after cerebral injury. It is considered that microglia progenitor cells in the adult brain may be lacking. The rapidly proliferated microglia after the injury are solely derived from residual microglia rather than microglia progenitors.
Functions of Microglia Were Heterogenous After Ischemic Stroke
It is widely accepted that targeting microglia activation could inhibit inflammatory injury and facilitate better stroke recovery. However, depletion of microglial cells exacerbates ischemic injury and dysregulates neuronal network activity. It is predicted that detrimental outcomes of excessive microglial activation after ischemia could be counterbalanced by beneficial outcomes, namely, phagocytosis and release of trophic factors. It also provides a hint that microglia demonstrate great heterogeneity, which calls for further investigations regarding microglia subtypes or specific functions. Upon the challenge of ischemic stroke, rest microglia can be activated in response to the inflammatory triggers and differentiates into two phenotypes like macrophage:M1 and M2. M1 macrophages (classically activated) are thought to be neurotoxic and are related to the production of proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), while M2 macrophages (alternatively activated) are neuroprotective and promote tissue repair and stroke recovery.
It is worth mentioning that M2 microglia are proven to show the ability of phagocytosis and their capability of cleaning neuronal debris reduces brain damage after stroke. In vitro assays suggest that IL-10-induced M2 microglia present enhanced phagocytosis abilities. However, this classification method could not adequately reflect the complicated microglial characteristics in the activated state. In recent studies, researchers even failed to find pure M1 or M2 state in vivo. With the development of single cellsequencing techniques, scientists prefer to designate the microglia profile regarding its unique functions in specific diseases. For instance, one unique microglia population with the potential to restrict neurodegeneration of Alzheimer’s disease is name neurodegenerative microglia (DAM). Therefore, we also expect to characterize microglia in ischemic stroke from a functional perspective.
Microglia Invade Infarcted Areas Earlier Than Macrophages
Due tosimilar histological phenotypes, the contributionofactivated resident microglia or infiltrated macrophages to stroke pathology has been difficult to distinguish. On Day 1 after focal cerebral ischemia, themicroglia amount remains unchanged. However, they switch their morphology into an ameboid and rounded shape with acquiring phagocytic properties andphagocytose neuronal cell debris. Thereafter, the microglia continue to proliferate during the first two weeks and show the most powerful phagocytic capacity within the first 2 days ofstroke, which proceeds and predominates overphagocytes ofhematogenous origin . Similarly, histological studies ofgreen fluorescent protein (GFP) transgenic bonemarrow chimeric mice with transient or permanent stroke models pointed that blood-derivedmacrophages infiltrated infarct areawith a delay ofat least 24 to 48 h after stroke onset.
Taken together, studies suggest that macrophages invade the ischemic site ofstroke later than microglia. The microglia are the main phagocytes during the first three days after stroke.In contrast to the rapid microglial response, the macrophages are rarely detected during the first 48 h and then gradually increase with the peak time in the first week after stroke .Transcriptomic studies of macrophages at Day 5 post stroke showed that infiltrated macrophages promote efferocytosis and inflammation resolution after ischemic stroke.
ENGULFMENT PROPERTIES OF MICROGLIA AFTER STROKE
Whether the phagocytic property of microglia in stroke is harmful or beneficial remains unclear. For instance, some researchers found that upregulating phagocytic capability of microglia in transient middle cerebral artery occlusion (tMCAO) mouse model promoted efficient clearance of tissue debris, facilitated tissue reconstruction, and reorganized neuronal network. Conversely, other scholars suggested that bexcessively activated microglia engulfed live or stressed neuronal cells in endothelin-1 induced focal cerebral ischemia model, which resulted in function deficits and brain atrophy after stroke. Therefore, microglial phagocytosis after stroke is a very interesting and complicated biological progress that may depend on the severity of initial ischemia, the advent or not of reperfusion, the location within the lesion (core or penumbra as defined afterwards), and the considered time points post-ictus. In addition, different experimental techniques and stroke models could be one reason for result discrepancy. Therefore, we summarized the various techniques used to measure phagocytic capability in Table 1.
Another critical reason is that the comprehensive phagocytic effect ofmicroglia may depend on their swallowing different cells mediated by specific “eat-me” signaling pathways. Understanding these mechanisms of microglia-mediated engulfment upon ischemic injury may open up exciting new therapeutic avenues for combating acute stage inflammation and late-stage synaptic refinements.
另一个关键原因是小胶质细胞的综合吞噬作用可能依赖于它们吞噬由特定的“吃我”信号通路介导的不同细胞。了解缺血性损伤时小胶质细胞介导的吞噬机制可能为对抗急性炎症和晚期突触精细化开辟新治疗途径。
Stroke triggers a cascade of events leading to rapid neuron and oligodendrocyte injury in the infarcted core (unsalvageable infarcted tissue with rCBF values less than 10 ml/100 g/min), and also in the penumbra area (salvageable infarcted tissue with rCBF values of 10 to 22 ml/100 g/min). Initially, stressed neurons or oligodendrocytes release damage associated molecular patterns (DAMPs), which act as eat-me signals attracting microglial phagocytosis. Subsequently, severe or prolonged ischemic injury produces a large number of cell debris. Efficient clearance of cell debris by microglia could be positive feedback for neurogenesis and initiate stroke recovery. Additionally, microglial phagocytosis may destabilize blood brain barrier (BBB) integrity by engulfing endothelial cells and regulate the inflammatory response by engulfing polymorphonuclear neutrophil granulocytes (PMNs). We here summarized these biological properties.
The Engulfment of Neurons
It is beneficial in part that microglia are capable of rapidly clearing dead or dying neurons within hours after stroke. Therefore, microglia play a fundamental role in facilitating the reorganization of neuronal circuits and resolving inflammation, especially in the ischemic core. Nevertheless, in areas of penumbra, stressed but alive neurons expose an eat-me signal called phospholipid phosphatidylserine (PS) in a reversible manner. Phagocytosis of these stressed but viable neurons is harmful to recovery, causing brain atrophy and motor dysfunction, which is also defined as phagoptosis. Communication between microglia and stressed neurons is interesting. Upon ischemia, stressed neurons express TMEM16F, which mediates the exposure of PS. TMEM16F knockdown blocked microglial phagocytosis of viable neurons in the penumbra and improved functional recovery in rat MCAO model. On the other hand, to resist phagoptosis, neurons may transfer microRNA-98 to microglia via extracellular vesicle secretion to prevent the salvageable neurons from microglial phagocytosis in tMCAO model.
The Cleaning of Myelin
In the research on remyelination of multiple sclerosis, it was found that the proliferation of oligodendrocyte progenitor cells (OPCs) and their migration to the lesion are not pivotal players in remyelination, while the differentiation of OPCs is more critical for remyelination. The debris from injured myelin and death cells suppress differentiation of OPCs . Moreover, the mononuclear phagocytic system mediates debris phagocytosis, improves the immune microenvironment of OPCs differentiation, and plays a necessary role in the restoration of white matter damage. Although white matter injury occupies nearly half of ischemic infarct volume, few studies are concerned about microglia mediated myelin clearance in ischemic stroke. Pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, exerts neuroprotective effects against ischemic stroke in a way that accelerates the phagocytosis of myelin debris by microglia. Mechanically, metabolic analysis showed that the clearance of cholesterol-rich myelin debris by microglia could synthesize desmosterol and activate liver X receptor (LXR) signaling, which resolves inflammation and creates a favorable environment for oligodendrocyte differentiation . Whether this mechanism is applicable to
stroke model remains to be studied.
The Cleaning of Cell Debris
Microglia, especially M2 microglia, have the constant ability to clear cell debris in ischemic stroke and consequently attenuate the detrimental effects ofinflammation. Debris clearance efficiency by microglia could be affected by the local microenvironment. For example, a positive correlation was found between the TNF expression level and phagocytic
activity in stroke-lesioned rodent brain. The ability of microglia to phagocytize and clean cell debris can be halted by reactive oxygen species (ROS) including superoxide (O2−).
细胞碎片的清洁
小胶质细胞，特别是M2小胶质细胞，具有持续清除缺血性卒中细胞碎片的能力，从而减轻了炎症的有害影响。小胶质细胞清除碎片的效率可能受到局部微环境的影响。例如，脑卒中病变啮齿动物的TNF表达水平与吞噬活性呈正相关。小胶质细胞吞噬和清洁细胞碎片的能力可以通过包括超氧化物（O2−）在内的活性氧（ROS）停止。
In addition to controlling detrimental inflammation,microglial phagocytosis does a remarkable job of promoting neurogenesis through clearing debris. Neurogenesis takes place in regions like cerebral neocortex, subventricular zone (SVZ), and sub-granular zone (SGZ), which happens immediately after brain ischemic challenge. Microglia isolated from the SVZ supported neurosphere generation in vitro, indicating the supporting role of microglia in neurogenesis in SVZ. Studies demonstrated that prefrontal stroke disturbed homeostasis of microglial phagocytosis, presenting with accumulating apoptotic cells in the SGZ. In early postnatal rats subjected to hemisphere ischemia, microglia accumulated and displayed engulfment properties in the ipsilateral SVZ region, while in adult rat stroke brain, it is reported that activated microglia increased in ipsilateral SVZ region concomitant with neuroblast migration into the ischemic region Even though knowledge about the effects of microglial phagocytotic populations in SVZ region of ischemia is still scarce, the phagocytic nature of SVZ microglia in adult has been reported. Enhancement of phagocytosis by sevoflurane treatment might lead to a favorable microenvironment for neuroblast survival. Consistently, intraperitoneal injection of minocycline (amicroglial inhibitor) hampered the activation of microglia and obstructed neurogenesis at the same time .
The Engulfment of Polymorphon-uclearNeutrophil Granulocytes (PMNs)
PMNs infiltrate the brain parenchyma 1 day after focal ischemia, which induces inflammation and exacerbates neuronal damage through releasing oxygen radicals, proteases, and proinflammatory cytokines . Thus, the prevention of infiltration and the reduction of the number of PMNs are putatively protective. Applying invading PMNs into organotypic brain slices enhances ischemic neurotoxicity, which could be counteracted by additional application of microglia. Several recent studies have shown that microglia can engulf these infiltrating neutrophils. In hippocampal slice cultures exposed to oxygen glucose deprivation, microglia engulfapoptotic PMNs and viable, motile, non-apoptotic PMNs, adopting a “chasing behavior” by time- lapse image . In the rat ischemic brain, the number of neutrophils is controlled by microglial phagocytosis. Two photon microscopy intuitively presented neutrophils which infiltrated into the ischemic brain parenchyma cross talked with microglia and were engulfed by them. The engulfment of PMNs may prevent the release ofneurotoxic compounds because dying PMNs secrete toxic compounds and also living PMNs.As such, scavenging PMNs alleviate neuron injury by converting the microenvironment from pro-inflammatory to anti-inflammatory.
The Engulfment of Vascular Endothelial Cells
Using two-photon microscopy and CX3CR1+/GFP mice, microglia were detected to expand toward adjacent blood vessels within 24 h post tMCAO (33). Subsequently, these perivascular microglia started to eat up endothelial cells by phagocytosis, which caused the disintegration of blood vessels with an eventual breakdown of the BBB. Accordingly, loss-of-microglia-function studies displayed a reduction in the extravasation of contrast agent into the brain penumbra and a decreased infarct size as measured by MRI. Engulfment properties of microglia and their potential pathological outcome after ischemic stroke are depicted in Figure 1.
SIGNALING MOLECULES RESPONS-IBLE FOR MICROGLIA PHAGOCYTOSIS AFTER STROKE
Professional phagocytes constantly fulfill a monitoring role rely on immune molecules to identify targeted pathogens or debris in need ofremoval. All phagocytic behaviors start with the exposure of “eat-me” signals from the target cells or debris. By sensing exposed “eat-me” signals, microglia begin rapid recognition and engulfment of target cells. PS is the best-known “eat-me” signal. Once exposed on the cell surface, it will modulate a crucial process of phagocytosis. PS appears usually inside of the cellular membrane and translocates to the external side of membrane under various circumstances, such as oxidative stress, inflammation, and growth-factor withdrawal. Apart from PS, calreticulin and desialylated cell surface glycoproteins also act as “eat-me” signals. Calreticulin is normally localized in the endoplasmic reticulum and translocates on the cell surface as a result of endoplasmic reticulum stress . The chemistry process of desialylation is catalyzed by sialidases (also known as neuraminidases). Sialidases integrate on cytomembrane and remove sialic acid residue after cell stress. Opsonins are soluble proteins binding the “eat-me” signal to its corresponding receptor on professional phagocytes, which are engaged subsequent to “eat-me” signal exposure. As an illustration,galectin-3 (Gal-3) is one of the opsonins secreted by macrophages and microglia. Upon LPS stimulation, Gal-3 is rapidly released from activated microglia and binds to Mertk on microglial cells and also desialylated sugar chains of stressed or dying neuronal cells. Multiple soluble molecules, namely, growth arrest-specific protein 6 (Gas6) or Protein S, milk fat globule EGF factor 8 (MFG-E8) opsonizes PS-exposing cells or other eat-me signals and is subsequently recognized by unique receptors on professional phagocytes. Here, we summarized emergent eat-me/opsonin/phagocytic receptor systems in
ischemic stroke in Figure 2.
TAM Receptors
TAM is an acronym for Tyro3, Axl, and Mer (protein designation Mer, c-Mer, or Mertk), which is widely expressed on the phagocyte surface of central nervous, immune, vascular, mammalian reproductive system, and retina. Mice with triple mutations in Tyro3, Axl,and Mer display a severe lymphoproliferative disorder accompanied by broad-spectrum autoimmunity. In the human brain, Axl is highest in mature astrocytes, microglia, oligodendrocytes, endothelial cells, and neurons. Mer expression is highest in both microglia and mature astrocytes, still detected in oligodendrocytes. Tyro3 is expressed on mature oligodendrocytes and has been implicated in multiple sclerosis susceptibility and myelin production.
Protein S is a vitamin K-dependent anticoagulant plasma glycoprotein. Growth arrest-specific protein 6 (Gas6), a structural analog of protein S, acts as a molecular bridge between the TAM receptors and eat-me signals together with protein S. In retinal pigment epithelial cells, either Mer mutation or concerted deletion of Gas6/protein S ligands disrupts circadian phagocytosis of photoreceptor outer segments. As mentioned above, Gal-3 has also been identified as a Merligand.
The pathological roles of TAM receptors in ischemic stroke have begun to be addressed directly. Protein S blocks endothelial injury and MCAO-induced BBB leakage after ligation of Tyro through activating sphingosine 1-phosphate receptor other than Mer or Axl. Likewise, protein S obstructs the extrinsic apoptotic cascade through Tyro3-dependent phosphorylation of FKHRL1 which may reduce post-ischemic neuronal toxicity. Endogenous expression of Axl and Gas6 increased in microglia/macrophage after stroke, while intranasal injection of recombinant Gas6 (rGas6) reduced the neurological deficits through inhibiting neuroinflammation by inhibiting TLR/ TRAF/NF-kappaB pathway.
Upon PS exposure, another opsonin molecule MFG-E8 and its microglial receptors vitronectin receptors (VNRs) mediate phagocytosis through activating a CRKII–DOCK180–RAC1 signaling pathway, which results in remodeling of microglial cytoskeleton Previous findings confirmed that microglia express Mer and MFG-E8 in a high-level response to inflammatory stimuli after ischemia stroke. Compared to wild type mice, mice lacking Mer or MFG-E8 appeared a decreased loss of viable neurons after brain ischemia stroke, which leads to a great improvement of motor function recovery . Molecular signaling pathways regarding TAM receptors are summarized in Figure 2A.
C1q and C3 Complement System
The complement components C3 and C1q may induce phagocytosis by binding to dying cell surfaces. C3 is activated and cleaved into the small protein C3a and the larger C3b complex by C3 convertase. C3a could attract immune cells and modulate the immune response by interacting with the cellular receptor C3a receptor (C3aR). C3b along with its cellular receptor CR3 mediates the clearance of dying cells and modulates the adaptive immune response.
C1q, the biggest protein of the C1 complex, is present in the neutrophils, microglia, and a subset of interneurons. Interestingly, C1q can enhance microglial clearance of apoptotic cells independent of C1r and C1s after ischemic stroke. On the intact cell surface, sialic acid modification of glycoproteins or glycolipids can act as a “don’t-eat-me” signal by preventing complement C3b and C1q binding. On the dying cell surface, glycoproteins or glycolipids could be desialylated. Then, C1q mediates phagocytic recognition by means of binding to desialylated glycoproteins. Apart from desialylated glycoprotein, calreticulin also acts as an opsonin for C1q. When C1q opsonizes dead cells, it could not only mediate engulfment by binding to lipoprotein receptor-related protein (LRP) on microglia, but also promote the conversion of C3 to C3b and trigger C3b-based phagocytosis.
In the developing CNS, C1q and C3 have been identified as important factors for controlling synaptic pruning. Microglia
engulf presynaptic inputs during peak retinogeniculate pruning is dependent on C3b/CR3 signaling pathway. After ischemic stroke, complement activation directs continuous microglia-dependent phagocytosis of hippocampal synapses and penumbral salvageable neurons, leading to cognitive decline. B4Crry is a targeted complement inhibitor inhibiting all complement pathways at the central C3 activation step. B4Crry prevents phagocytosis of penumbral neurons and hippocampal synapses, improving long-term motor and cognitive recovery. C3aR antagonist, SB 290157 given intracortically, may limit neuroinflammation and neuronal death after ischemia by restraining microglia transition to the phagocytic type. In chronic cerebral hypoperfusion rats, SB290157 decreased the number ofmicroglia adhering to myelin, attenuated white matter injury and cognitive deficits . Generally, animal studies suggest that activation of the C1q/C3 system after stroke may be detrimental to recovery.
Molecular signaling pathways regarding complement system are summarized in Figure 2B.
Triggering Receptor Expressed on Myeloid Cells-2 (TREM2)-Activating Protein of 12 kDa (DAP12) System
TREM2 was originally described on circulating macrophages, where it is bound to anionic moieties on exogenous pathogens and mediated pathogen clearance. DAP12 is an intracellular membrane adaptor of TREM2. Accumulating evidence demonstrated that TREM2-DAP12 signaling is involved in microglial phagocytosis. Human beings with a loss function of either TREM2 or DAP12 develop an inflammatory neurodegenerative disease—Nasu-Hakola disease (NHD),leading to death in the fourth or fifthdecadeoflife. TREM2 deficiency in microglia inhibits apoptotic neuronal clearance and increases the production of inflammatory mediators such as TNF-a.
髓样细胞表达触发受体-2（TREM 2）-12 kDa激活蛋白（DAP 12）系统
TREM 2最初在循环巨噬细胞上描述，其中其与外源性病原体上的阴离子部分结合并介导病原体清除。DAP 12是TREM 2的细胞内膜衔接子。越来越多的证据表明，TREM 2-DAP 12信号转导参与小胶质细胞的吞噬作用。TREM 2或DAP 12功能丧失的人会发展成炎性神经退行性疾病-纳苏-哈科拉病（NHD），导致40或50岁的死亡。小胶质细胞中的TREM 2缺陷抑制凋亡神经元清除并增加炎症介质如TNF-α的产生。
TREM2 participates in phagocytic activity following experimental stroke. TREM2 deficiency can lead to worsened outcomes after ischemic stroke by decreasing the phagocytosis of injured neurons. With the application of bone marrow chimeric mice, this team further addressed that intact microglia TREM2 is more important for beneficial phagocytosis and stroke recovery than that of circulating macrophage. Additionally, another research group reported that TREM2 could promote a microglial switch from the detrimental M1 phenotype to the beneficial M2 phenotype, which may affect the short-term outcome in the mouse MCAO model. Ligand or opsonin ofTREM2 was less reported. It is predicted that the potential endogenous binding partner of TREM2 in ischemic brain is probably high-molecular-weight nucleic acids released by damaged cells. In conclusion, the TREM2-DAP12 signaling is mainly considered beneficial in ischemic stroke. Targeting TREM2 signaling especially in microglia has become a therapeutic target as it was shown that the systemic administration of a TREM2 agonist or TREM2 overexpression had a neuroprotective effect in ischemic injury.
Molecular signaling pathways regarding TREM2-DAP12 system are summarized in Figure 2C.
Nucleotides and P2 Metabolic Purinoceptors
P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2Y containing eight types (P2Y1, 2, 4, 6, 11, 12, 13, and 14) are activated by nucleotides and couple to intracellular second messenger systems through heteromeric G-proteins. P2Y6 receptor is actively responsive to UDP and partially responsive to UTP and ADP. It has been demonstrated that microglial purinergic P2Y6 receptor is activated by UDP released from damaged neurons and triggers microglial phagocytosis. In other words, UDP, which is released from injured neurons after trauma or ischemia, acts as an “eat-me” signal and meditates the P2Y6-dependent phagocytosis. P2Y6 is combined with UDP, and then activates phospholipase C (PLC) which in turn causes the synthesis of inositol 1,4,5-trisphosphate (InsP3) and triggers the booted release ofCa2+ from InsP3-receptor-sensitive stores. On top of triggering the intracellular Ca2+ over-loading, the P2Y6-receptor-signaling pathway triggers actin cytoskeleton polarization to shape filopodia-like protrusions, thus facilitating the engulfment of cell debris
The research newly reported that the expression of the P2Y6 receptor in microglia increased after tMCAO and P2Y6 receptor antagonist MRS2578 treatment inhibited microglia to swallow apoptosis cell debris, subsequently aggravating neurological function. The possible mechanism of P2Y6 receptor-mediated phagocytosis is related to myosin light chain kinase After all, it indicates that P2Y6/UDP-mediated microglial phagocytosis plays a favorable part in the acute stage of ischemic stroke, which can be a therapeutic target for ischemic stroke.
Apart from P2Y6/UDP signaling pathway, neuronal injury gives rise to the leakage of ATP or ADP that appears to be a “find-me” signal to attract microglia and cause chemotaxis through P2Y12 receptors. In the hippocampus regions of young mice, the percentage of apoptotic cell engulfment significantly reduces in P2Y12 deficient mice, which impacts the neurogenesis of hippocampus SGZ. Following cerebrovascular damage, P2Y12-mediated chemotaxis of microglia is central to the maintenance of BBB integrity.
The P2Y6/UDP and P2Y12/ADP signaling pathways are depicted in Figure 2D.
研究新报道tMCAO后小胶质细胞P2 Y 6受体表达增加，P2 Y 6受体拮抗剂MRS 2578处理抑制小胶质细胞吞噬凋亡细胞碎片，从而加重神经功能。P2 Y 6受体介导的吞噬作用的可能机制与肌球蛋白轻链激酶有关。提示P2 Y 6/UDP介导的小胶质细胞吞噬功能在缺血性脑卒中急性期发挥了积极作用，可作为缺血性脑卒中的治疗靶点。
除了P2 Y 6/UDP信号传导途径，神经元损伤引起ATP或ADP的泄漏，这似乎是一种“找到我”信号，吸引小胶质细胞并通过P2 Y12受体引起趋化性。在幼年小鼠的海马区，P2 Y12缺陷小鼠的凋亡细胞吞噬百分比显著降低，这影响了海马SGZ的神经发生。脑血管损伤后，P2 Y12介导的小胶质细胞趋化性是维持血脑屏障完整性的关键。
P2 Y 6/UDP和P2 Y12/ADP信号传导途径如图2D所示。
CD47-Signal Regulated Protein Alpha (SIRPa) System
Inhibitory signals can also modulate microglial phagocytosis, so-
called “don’t-eat-me” signals. In the immune system, SIRPa acts as a “don’t-eat-me” signal, which is required for optimal and appropriate microglial engulfment. CD47 is documented to express on synapses, oligodendrocytes, erythrocytes, and microglia. Its receptor SIRPa is an Ig superfamily protein and has been observed on microglia or macrophage in the CNS.The expression pattern of CD47-SIRPa is correlated with peak pruning in the developing retinogeniculate system and preventsexcess microglial phagocytosis according to literature. This
inhibitory engulfment property of CD47-SIRPa hindered the maintenance of myelin integrity following mild brain damage . In ischemic stroke, a study using CD47 knockout mice suggests that CD47 deletion reduces brain infarct and swelling at an acute stage in MCAO model through decreasing neuroinflammation. Notably, it is interpreted that CD47-deficient erythrocytes are more prone to be cleared by microglia/
macrophage. Injection of CD47 knockout blood into mouse brain resulted in quicker clot resolution and less brain swelling than WT blood. Consistently, CD47 blocking antibody speededuphematoma clearance and alleviated atherosclerosis through restoring phagocytosis by microglia/macrophage. Exempt from cerebral hemorrhage, these findings provide more clues for the treatment of hemorrhagi transformation in stroke patients. The CD47/SIRPa signaling pathway is depicted in Figure 2E.
In addition to the molecules described above, several other phagocytic molecules are still being reported and studied, although their functions in ischemic stroke are unknown. For instance, CD22 blocks microglia-mediated engulfment of myelin debris in the aged brain, while CD22 blockade restores microglial homeostasis and cognitive impairment in aged mice.
RXR/PPAR-g is a critical transcription factor in the nuclear
receptor superfamily and is responsible for the expression of scavenger receptors. Mice lacking RXR-a in myeloid phagocytes demonstrated worsened late functional recovery and serious brain atrophy in MCAO dependent model.
We still have a long way to thoroughly understand the
molecular pathways of phagocytosis. We expect to find specific phagocytosis pathways that protect ischemic brain injury without causing excessive alive neuronal clearance in the future.
CD 47-信号调节蛋白α（SIRPa）系统
抑制信号也能调节小胶质细胞吞噬作用，所以叫做“别吃我”信号。在免疫系统中，SIRPa充当“不吃我”的信号，这是最佳和适当的小胶质细胞吞噬所必需的。CD 47被记录在突触、少突胶质细胞、红细胞和小胶质细胞上表达。其受体SIRPa是一种Ig超家族蛋白，在CNS的小胶质细胞或巨噬细胞上被观察到。CD47-SIRPa的表达模式与发育中的视网膜生成系统的峰值修剪和预防性小胶质细胞吞噬有关。这一点CD47-SIRPa的抑制性吞噬特性阻碍了轻度脑损伤后髓磷脂完整性的维持。在缺血性卒中中，一项使用CD 47基因敲除小鼠的研究表明，在MCAO模型中，通过减少神经炎症，CD 47缺失可减少脑梗塞和急性期肿胀。值得注意的是，CD47缺陷型红细胞更容易被小胶质细胞清除。巨噬细胞.CD 47基因敲除血液注入小鼠大脑后，血块分辨率比WT血液更快，脑肿胀更少。CD 47通过小胶质细胞/巨噬细胞恢复吞噬作用，持续阻断抗体快速血肿清除，缓解动脉粥样硬化。除脑出血外，这些发现为卒中患者治疗出血性转化提供了更多线索。图2E说明了CD47/SIRPa信号通路。
除了上述分子外，其他几种吞噬细胞分子仍在报道和研究中，尽管它们在缺血性卒中中的功能尚不清楚。例如，CD22阻断小胶质细胞介导的老年脑髓鞘碎片的吞噬，而CD22阻断可恢复老年小鼠的小胶质细胞稳态和认知障碍。
RXR/PPAR-g是核受体超家族中的关键转录因子，负责清道夫受体的表达。在骨髓吞噬细胞中缺乏RXR-α的小鼠在MCAO依赖性模型中表现出恶化的晚期功能恢复和严重的脑萎缩。
我们仍然有很长的路要走，彻底了解吞噬作用的分子途径。我们期望在未来找到保护缺血性脑损伤而不引起过度活神经元清除的特异性吞噬途径。
OTHER IMMUNE CELLS CONTRIBUTING
TO PHAGOCYTOSIS IN ISCHEMIC
STROKE
The lack of precise discriminating markers between the myeloid populations (macrophage and microglia) has led many studies
summarized above to generate conclusions based on the grouping of the two populations. In addition to microglia and macrophage, some other cells, namely, astrocytes, peripheral infiltrated PMNs, monocytes, and pericytes have been reported to present engulfment properties as well.
It was reported that monocyte infiltration occurs as early as 4 h after stroke. Infiltrating monocytes are primarily involved in early debris clearance and have significantly higher phagocytic capacity at 72 h after stroke compared with microglia. An earlier study underscored the importance of monocytes to hemorrhage clearance in a model of intracerebral hemorrhage and highlighted their ability to significantly improve long-term outcomes through phagocytic function early after stroke. Cell surface scavenger receptor CD36 on monocyte-derived macrophages mediates phagocytosis during the recovery phase in post-stroke brains and plays a reparative role during the resolution of inflammation in ischemic stroke.
其他免疫细胞参与缺血性卒中的吞噬作用
由于骨髓细胞群（巨噬细胞和小胶质细胞）之间缺乏精确的区分标志物，导致上文总结的许多研究基于两个群体的分组得出结论。除小胶质细胞和巨噬细胞外，据报道，一些其他细胞，即星形胶质细胞、外周浸润的PMN、单核细胞和周细胞也具有吞噬特性。
据报道，单核细胞浸润发生早在卒中后4小时。浸润的单核细胞主要参与早期碎片清除，与小胶质细胞相比，在卒中后72小时具有显著更高的吞噬能力。一项早期研究强调了单核细胞在脑出血模型中清除出血的重要性，并强调了其在卒中后早期通过吞噬功能显著改善长期预后的能力。单核细胞衍生的巨噬细胞上的细胞表面清道夫受体CD36在卒中后脑的恢复阶段介导吞噬作用，并在缺血性卒中的炎症消退过程中发挥修复作用。
PMNs elicit rapid immune responses compared with mononuclear phagocytes. The infiltration of neutrophils can be detected within 3 h after the onset of ischemic stroke. One subtype of PMNs named N2 phenotype represents a proresolving function by producing anti-inflammatory factors or
engulfing cellular debris. A further study corroborates that the N2 phenotype facilitates neutrophil clearance by macrophage and does not induce neuronal death after ischemic injury. Meanwhile, skewing neutrophils toward the N2 phenotype before stroke reduced infarct volumes at 1 day after MCAO.
As BBB components, pericytes reside in the abluminal side of endothelial cells lining the capillaries in the brain. Pericytes are considered to be multipotent progenitor cells and exhibit microglia-like properties after ischemia insult. In response to ischemic insult, pericytes could leave blood vessel wall and migrate into the ischemic brain parenchyma, where they express microglia-specific markers, namely, Iba-1 and gal-3. In human stroke postmortem sections, a portion of gal-3 positive pericytes were found in the peri-infarct area. Platelet-derived growth factor receptor-b (PDGFR-b) is a specific marker of pericytes. PDGFR-b+ pericytes, isolated from ischemic brain tissue, could differentiate into Iba-1+ ameboid-like microglia and own the capability to phagocytose latex beads.
与单核吞噬细胞相比，中性粒细胞引起快速的免疫应答。中性粒细胞浸润可在缺血性卒中发作后3小时内检测到。一种称为N2表型的中性粒细胞亚型通过产生抗炎因子或吞噬细胞碎片而具有促消退功能。进一步的研究证实，N2表型促进巨噬细胞对中性粒细胞的清除，并且在缺血性损伤后不诱导神经元死亡。同时，卒中前中性粒细胞偏向N2表型减少了MCAO后1天的梗死体积。
作为血脑屏障的组成部分，周细胞位于脑内毛细血管内皮细胞的近腔侧。周细胞被认为是多能祖细胞，在缺血损伤后表现出小胶质细胞样特性。在缺血性损伤的反应中，周细胞可以离开血管壁并迁移到缺血性脑实质中，在那里它们表达小胶质细胞特异性标记物，即Iba-1和gal-3。在人类卒中尸检切片中，在梗死周围区域发现了一部分gal-3阳性周细胞。血小板衍生生长因子受体-b（PDGFR-b）是周细胞的特异性标志物。从缺血性脑组织中分离的PDGFR-b+周细胞可分化为Iba-1+阿米巴样小胶质细胞，并具有吞噬乳胶珠的能力。
It has been recently observed that astrocytes, acting like nonprofessional phagocytes, can also contribute to the elimination of apoptotic neurons, synapses, and cell debris after transient focal ischemia in mice. Astrocytes become phagocytic
during the late stage of ischemia, and ABCA1 along with its downstream molecules plays a pivotal role in this process. Phagocytic astrocytes have a distinct role from microglia given that phagocytic astrocytes were observed in the ischemic penumbra region in the late phase of stroke, while phagocytic
microglia were located in the infarcted core in the early stage of stroke. Judging from the spatiotemporal pattern of astrocytic phagocytosis after stroke, it is predicted that
astrocytes would be involved in the elimination of debris and synapses, thereby leading to repairing/remodeling of the
ischemic penumbra region.
最近观察到，星形胶质细胞与非专职吞噬细胞一样，也可在小鼠短暂局灶性缺血后消除凋亡神经元、突触和细胞碎片。星形胶质细胞在缺血晚期成为吞噬细胞，ABCA 1沿着其下游分子在此过程中起关键作用。吞噬性星形胶质细胞的作用与小胶质细胞不同，因为在卒中晚期的缺血性半影区域观察到吞噬性星形胶质细胞，而在卒中早期，吞噬性小胶质细胞位于梗死核心。从卒中后星形胶质细胞吞噬作用的时空模式判断，预测星形胶质细胞将参与碎片和突触的消除，从而导致缺血性半影区域的修复/重塑。
Nevertheless, resident microglia, astrocyte, monocyte/macrophage system, peripheral neutrophils, and pericytes may cooperatively modulate the removal of debris and synapse
architecture after stroke.
CONCLUDING REMARKS
Microglial phagocytosis is a double-sword to immunoinflammation and stroke recovery. Microglia engulf live neurons and endothelial
cells, which results in excessive neuronal death and BBB leakage respectively. On the other hand, microglia restrict inflammatory
damage via engulfing cell debris, cleaning infiltrating neutrophils, and creating an optimal microenvironment for neurogenesis.
Therefore, understanding the molecular mechanisms and modulating selective microglial phagocytosis represent an attractive target with a long therapeutic window after stroke in the future.
然而，驻留的小胶质细胞，星形胶质细胞，单核细胞/巨噬细胞系统，外周中性粒细胞，周细胞可以协同调节卒中后的碎片和突触结构的去除。
结论
小胶质细胞吞噬作用是免疫炎症和卒中恢复的双刃剑。小胶质细胞吞噬活的神经元和内皮细胞，这分别导致过度的神经元死亡和BBB渗漏。另一方面，小胶质细胞通过吞噬细胞碎片、清洁浸润的中性粒细胞和为神经发生创造最佳微环境来限制炎性损伤。因此，了解分子机制和调节选择性小胶质细胞吞噬作用是未来卒中后具有长治疗窗的有吸引力的目标。
结束语
神经内科3病区全体，预祝大家万事顺利、心想事成！