Brain plasticity and microglia: is transsynaptic glial activation in the thalamus after limb denervation linked to cortical plasticity and central sensitisation?

Microglia are a subset of tissue-macrophages that are ubiquitously distributed throughout the entire CNS. In health, they remain largely dormant until activated by a pathological stimulus. The availability of more sensitive detection techniques has allowed the early measurement of the cell responses of microglia in areas with few signs of active pathology. Subtle neuronal injury can induce microglial activation in retrograde and anterograde projection areas remote from the primary lesion focus. There is also evidence that in cases of long-standing abnormal neuronal activity, such as in patients after limb amputation with chronic pain and phantom sensations, glial activation may occur transsynaptically in the thalamus. Such neuronally driven glial responses may be related to the emergence central sensitisation in chronic pain states or plasticity phenomena in the cerebral cortex. It is suggested, that such persistent low-level microglial activation is not adequately described by the traditional concept of phagocyte-mediated tissue damage that largely evolved from studies of acute brain lesion models or acute human brain pathology. Due to the presence of signal molecules that can act on neurons and microglia alike, the communication between neurons and microglia is likely to be bi-directional. Persistent subtle microglial activity may modulate basal synaptic transmission and thus neuronal functioning either directly or through the interaction with astrocytes. The activation of microglia leads to the emergence of microstructural as well as functional compartments in which neurokines, interleukins and other signalling molecules introduce a qualitatively different, more open mode of cell-cell communication that is normally absent from the healthy adult brain. This 'neo-compartmentalisation', however, occurs along predictable neuronal pathways within which these glial changes are themselves under the modulatory influence of neurons or other glial cells and are subject to the evolving state of the pathology. Depending on the disease state, yet relatively independent of the specific disease cause, fluctuations in the modulatory influence by non-neuronal cells may form the cellular basis for the variability of brain plasticity phenomena, i.e. the plasticity of plasticity.     

Hypoxia/Reoxygenation induces nitric oxide and TNF-alpha release from cultured microglia but not astrocytes of the rat

Hypoxia/reoxygenation (H/R) elicits neuronal cell injury and glial cell activation within the central nervous system (CNS). Neuroinflammation is a process that primarily results from the acute or chronic activation of glial cells. This overactive state of glial cells results in the increased release of nitric oxide (NO) and/or tumor necrosis factor alpha (TNF-alpha), a process which can lead to neuronal damage or death. In this study, we found that hypoxia for eight or twelve hours (h) followed by 24 h reoxygenation (H8/ R24 or H12/R24) induced NO production and TNF-alpha release from cultures of enriched microglial or mixed glial cells. However, microglial cells could not survive longer periods of hypoxia (> or = 12 h) in microglia-enriched culture. While astrocytes retained a 95% viability following longer periods of H/R in astrocyte-enriched cultures, they did not produce any significant quantities of NO and TNF-alpha. Reoxygenation for prolonged periods (three and five days) following H24 resulted in progressively greater increases in NO production (about two-fold greater level in hypoxia as compared to normoxic conditions) accompanied by relatively less increases in TNF-alpha release in mixed glial cell cultures. Our data indicate that inflammatory mediators such as NO and TNF-alpha are released from glia-enriched mix culture in response to H/R. While microglial cells are more vulnerable than astrocytes during H/R, they survive longer in the presence of astrocyte and are the major cell type producing NO and TNF-alpha. Furthermore, the TNF-alpha release precedes NO production in response to a prolonged duration of reoxygenation following hypoxia for 24 h.     

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1^GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.     

Role of IL‐15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T‐cell infiltration

The release of inflammatory mediators from immune and glial cells either in the peripheral or CNS may have an important role in the development of physiopathological processes such as neuropathic pain. Microglial, then astrocytic activation in the spinal cord, lead to chronic inflammation, alteration of neuronal physiology and neuropathic pain. Standard experimental models of neuropathic pain include an important peripheral inflammatory component, which involves prominent immune cell activation and infiltration. Among potential immunomodulators, the T‐cell cytokine interleukin‐15 (IL‐15) has a key role in regulating immune cell activation and glial reactivity after CNS injury. Here we show, using the model of chronic constriction of the sciatic nerve (CCI), that IL‐15 is essential for the development of the early inflammatory events in the spinal cord after a peripheral lesion that generates neuropathic pain. IL‐15 expression in the spinal cord was identified in both astroglial and microglial cells and was present during the initial gliotic and inflammatory (NFκB) response to injury. The expression of IL‐15 was also identified as a cue for macrophage and T‐cell activation and infiltration in the sciatic nerve, as shown by intraneural injection of the cytokine and activity blockage approaches. We conclude that the regulation of IL‐15 and hence the initial events following its expression after peripheral nerve injury could have a future therapeutic potential in the reduction of neuroinflammation.     

Regulation of Microglial Phagocytosis by RhoA/ROCK-Inhibiting Drugs

Inflammation within the central nervous system (CNS) is a major component of many neurodegenerative diseases. The underlying mechanisms of neuronal loss are not fully understood, but the activation of CNS resident phagocytic microglia seems to be a significant element contributing to neurodegeneration. At the onset of inflammation, high levels of microglial phagocytosis may serve as an essential prerequisite for creating a favorable environment for neuronal regeneration. However, the excessive and long-lasting activation of microglia and the augmented engulfment of neurons have been suggested to eventually govern widespread neurodegeneration. Here, we investigated in a functional assay of acute inflammation how the small GTPase RhoA and its main target the Rho kinase (ROCK) influence microglial phagocytosis of neuronal debris. Using BV-2 microglia and human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA activation and microglial phagocytosis of neuronal cell fragments. Inhibition of the downstream effector ROCK with the small-molecule agents Y-27632 and Fasudil reduces the engulfment of neuronal debris and attenuates the production of the inflammatory mediator nitric oxide during stimulation with lipopolysaccharide. Our results support a therapeutic potential for RhoA/ROCK-inhibiting agents as an effective treatment of excessive inflammation and the resulting progression of microglia-mediated neurodegeneration in the CNS.     

Tumor necrosis factor receptor-associated death domain mediated neuronal death contributes to the glial activation and subsequent neuroinflammation in Japanese encephalitis

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and in disease state, little is known regarding how the neuronal death initiates the cascades of secondary neuroinflammation. We have exploited an experimental model of Japanese encephalitis to better understand how neuronal death following viral infection initiates microglial activation following Japanese encephalitis virus infection. We have earlier shown that the altered expression of tumor necrosis factor receptor-1 (TNFR-1) and TNFR associated death domain (TRADD) following Japanese encephalitis virus infection regulates the downstream apoptotic cascades. Here we have reported that silencing TRADD expression with small-interfering RNA reduced neuronal apoptosis and subsequent microglial and astroglial activation and release of various pro-inflammatory mediators. Our findings suggest that the engagement of TNFR-1 and TRADD following Japanese encephalitis virus infection plays a crucial role in glial activation also and influences the outcome of viral pathogenesis.     

Role of Cell Cycle Proteins in CNS Injury

Following trauma or ischemia to the central nervous system (CNS), there is a marked increase in the expression of cell cycle-related proteins. This up-regulation is associated with apoptosis of post-mitotic cells, including neurons and oligodendrocytes, both in vitro and in vivo. Cell cycle activation also induces proliferation of astrocytes and microglia, contributing to the glial scar and microglial activation with release of inflammatory factors. Treatment with cell cycle inhibitors in CNS injury models inhibits glial scar formation and neuronal cell death, resulting in substantially decreased lesion volumes and improved behavioral recovery. Here we critically review the role of cell cycle pathways in the pathophysiology of experimental stroke, traumatic brain injury and spinal cord injury, and discuss the potential of cell cycle inhibitors as neuroprotective agents. Special issue dedicated to Dr. Moussa Youdim.     

Role of the Cell Cycle in the Pathobiology of Central Nervous System Trauma

Up-regulation of cell cycle proteins occurs in both mitotic and post-mitotic neural cells after central nervous system (CNS) injury in adult animals. In mitotic cells, such as astroglia and microglia, they induce proliferation, whereas in post-mitotic cells such as neurons they initiate caspase-related apoptosis. We recently reported that early central administration of the cell cycle inhibitor flavopiridol after experimental traumatic brain injury (TBI) significantly reduced lesion volume, scar formation and neuronal cell death, while promoting near complete behavioral recovery. Here we show that in primary neuronal or astrocyte cultures structurally different cell cycle inhibitors (flavopiridol, roscovitine, and olomoucine) significantly reduce up-regulation of cell cycle proteins, attenuate neuronal cell death induced by etoposide, and decrease astrocyte proliferation. Flavopiridol, in a concentration dependent manner, also attenuates proliferation/activation of microglia. In addition, we demonstrate that central administration of flavopiridol improves functional outcome in dose-dependent manner after fluid percussion induced brain injury in rats. Moreover, delayed systemic administration of flavopiridol significantly reduces brain lesion volume and edema development after TBI. These data provide further support for the therapeutic potential of cell cycle inhibitors for the treatment of clinical CNS injury and that protective mechanisms likely include reduction of neuronal cell death, inhibition of glial proliferation and attenuation of microglial activation.     

Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli

The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non-immune-mediated pathological conditions, little is known about the expression of the cannabinoid system during immune-mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real-time PCR, found a 100-fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10-fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up-regulation of the CB2 receptor, we cultured microglial cells with combinations of gamma-interferon (IFN-gamma) and granulocyte) macrophage-colony stimulating factor (GM-CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10-fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2-arachidonylglycerol (2-AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up-regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune-induced inflammation.     

Glutamate excitotoxicity is involved in the induction of paralysis in mice after infection by a human coronavirus with a single point mutation in its spike protein

Human coronaviruses (HCoV) are recognized respiratory pathogens, and some strains, including HCoV-OC43, can infect human neuronal and glial cells of the central nervous system (CNS) and activate neuroinflammatory mechanisms. Moreover, HCoV-OC43 is neuroinvasive, neurotropic, and neurovirulent in susceptible mice, where it induces chronic encephalitis. Herein, we show that a single point mutation in the viral spike (S) glycoprotein (Y241H), acquired during viral persistence in human neural cells, led to a hind-limb paralytic disease in infected mice. Inhibition of glutamate excitotoxicity using a 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propranoic acid (AMPA) receptor antagonist (GYKI-52466) improved clinical scores related to the paralysis and motor disabilities in S mutant virus-infected mice, as well as protected the CNS from neuronal dysfunctions, as illustrated by restoration of the phosphorylation state of neurofilaments. Expression of the glial glutamate transporter GLT-1, responsible for glutamate homeostasis, was downregulated following infection, and GYKI-52466 also significantly restored its steady-state expression level. Finally, GYKI-52466 treatment of S mutant virus-infected mice led to reduced microglial activation, which may lead to improvement in the regulation of CNS glutamate homeostasis. Taken together, our results strongly suggest an involvement of excitotoxicity in the paralysis-associated neuropathology induced by an HCoV-OC43 mutant which harbors a single point mutation in its spike protein that is acquired upon persistent virus infection.     

Glia maintain follower neuron survival during Drosophila CNS development

While survival of CNS neurons appears to depend on multiple neuronal and non-neuronal factors, it remains largely unknown how neuronal survival is controlled during development. Here we show that glia regulate neuronal survival during formation of the Drosophila embryonic CNS. When glial function is impaired either by mutation of the glial cells missing gene, which transforms glia toward a neuronal fate, or by targeted genetic glial ablation, neuronal death is induced non-autonomously. Pioneer neurons, which establish the first longitudinal axon fascicles, are insensitive to glial depletion whereas the later extending follower neurons die. This differential requirement of neurons for glia is instructive in patterning and links control of cell number with axon guidance during CNS development.     

Microglia and neuronal cell death

Microglia, the brain's innate immune cell type, are cells of mesodermal origin that populate the central nervous system (CNS) during development. Undifferentiated microglia, also called ameboid microglia, have the ability to proliferate, phagocytose apoptotic cells and migrate long distances toward their final destinations throughout all CNS regions, where they acquire a mature ramified morphological phenotype. Recent studies indicate that ameboid microglial cells not only have a scavenger role during development but can also promote the death of some neuronal populations. In the mature CNS, adult microglia have highly motile processes to scan their territorial domains, and they display a panoply of effects on neurons that range from sustaining their survival and differentiation contributing to their elimination. Hence, the fine tuning of these effects results in protection of the nervous tissue, whereas perturbations in the microglial response, such as the exacerbation of microglial activation or lack of microglial response, generate adverse situations for the organization and function of the CNS. This review discusses some aspects of the relationship between microglial cells and neuronal death/survival both during normal development and during the response to injury in adulthood.     

Inflammation and apoptosis: linked therapeutic targets in spinal cord injury

The secondary cascade of cell death that follows central nervous system (CNS) injury or ischemia has long been considered a target for neuroprotective agents aimed at sparing tissue and function. Recently, several laboratories have shown remarkable protection and recovery of function in rodent models of spinal cord injury using treatments that target components of the CNS inflammatory response. The use of minocycline, an antibiotic that reduces microglial activation, antibody blockade of the CD95 (FAS) ligand and the blockade of glycosphingolipid-induced iNOS (inducible nitric oxide synthase) have recently been shown to reduce neuronal and glial apoptosis with concomitant improvement in neurological function, and appear to enhance the efficacy of cell transplantation strategies.     

Regulation and Function of Cyclic GMP-Mediated Pathways in Glial Cells

A large body of evidence supports a role for the NO-cGMP-protein kinase G pathway in the regulation of synaptic transmission and plasticity, brain development and neuroprotection. Circumstancial evidence implicates natriuretic peptide-stimulated cGMP formation in the same CNS functions. In addition to neurons, both cGMP-mediated pathways are functional in glial cells and an increasing number of reports indicate that they may control important aspects of glial cell physiology relevant to neuronal function. In this article we briefly review the regulation of cGMP formation in glial cells and summarize recent evidence indicating that cGMP-mediated pathways can play important roles in astroglial and microglial function in normal and diseased brain. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Regulation of microglial migration,phagocytosis, and neurite outgrowth by HO‐1/CO signaling

Clearance of infected and apoptotic neuronal corpses during inflammatory conditions is a fundamental process to create a favorable environment for neuronal recovery. Microglia are the resident immune cells and the predominant phagocytic cells of the CNS, showing a multitude of cellular responses upon activation. Here, we investigated in functional assays how the CO generating enzyme heme oxygenase 1 (HO‐1) influences BV‐2 microglial migration, clearance of debris, and neurite outgrowth of human NT2 neurons. Stimulation of HO‐1 activity attenuated microglial migration in a scratch wound assay, and phagocytosis in a cell culture model of acute inflammation comprising lipopolysaccharide (LPS)‐activated microglia and apoptosis‐induced neurons. Application of a CO donor prevented the production of NO during LPS stimulation, and reduced microglial migration and engulfment of neuronal debris. LPS‐activated microglia inhibited neurite elongation of human neurons without requiring direct cell–cell surface contact. The inhibition of neurite outgrowth was totally reversed by application of exogenous CO or increased internal CO production through supply of the substrate hemin to HO. Our results point towards a vital cytoprotective role of HO‐1/CO signaling after microglial activation. In addition, they support a therapeutic potential of CO releasing chemical agents in the treatment of excessive inflammatory conditions in the CNS. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 854–876, 2015     

Nitric oxide regulates antagonistically phagocytic and neurite outgrowth inhibiting capacities of microglia

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by inflammatory cellular mechanisms, mainly resulting from the activation of central nervous system (CNS) resident microglia. Under inflammatory conditions, microglia up‐regulate the inducible isoform of NOS (iNOS), leading to the production of high concentrations of the radical molecule nitric oxide (NO). At the onset of inflammation, high levels of microglial‐derived NO may serve as a cellular defense mechanism helping to clear the damaged tissue and combat infection of the CNS by invading pathogens. However, the excessive overproduction of NO by activated microglia has been suggested to govern the inflammation‐mediated neuronal loss causing eventually complete neurodegeneration. Here, we investigated how NO influences phagocytosis of neuronal debris by BV‐2 microglia, and how neurite outgrowth of human NT2 model neurons is affected by microglial‐derived NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute inflammation comprising lipopolysaccharide (LPS)‐activated microglia and apoptotic neurons. Chemical manipulations suggested that NO up‐regulates phagocytosis independently of the sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit neurite outgrowth of human neurons via the generation of large amounts of NO over effective distances in the millimeter range. Application of a NOS blocker prevented the LPS‐induced NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider functionally antagonistic microglial cell responses during pharmaceutic therapy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 566–584, 2016     

Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury

Peripheral nerve injury can induce spinal microglial/astrocyte activation. Substances released by activated glial cells excite spinal nociceptive neurons. Pharmacological disruption of glial activation or antagonism of substances released by activated glia prevent or reverse pain hypersensitivity. It is not known, however, what causes spinal cord glia to shift from a resting to an activated state. In an attempt to understand the potential role of monocyte chemoattractant protein-1 (MCP-1) in triggering spinal glial activation and its contribution to the development of neuropathic pain, we investigated the effect of peripheral nerve injury on MCP-1 expression in dorsal root ganglia (DRG) and the spinal cord, and established its temporal relationship with activation of spinal microglia and astrocytes. We observed that MCP-1 was induced by chronic constriction of the sciatic nerve in DRG sensory neurons, spinal cord motor neurons and in the superficial dorsal horn, ipsilateral to the injury. Neuronal MCP-1 induction was followed by surrounding microglial activation. After peaking at day 7 after injury, MCP-1 levels began to decline rapidly and had returned to baseline by day 150. In contrast, microglial activation peaked by day 14 and declined afterwards to reach a lower, yet significantly raised level beyond day 22 and remained increased until the end of the test period. Astrocyte activation became detectable later, progressed more slowly and also remained increased until the end of the test period, in parallel with a decreased nociceptive threshold. Our results suggest that neuronal MCP-1 may serve as a trigger for spinal microglial activation, which participates in the initiation of neuropathic pain. Delayed, sustained astrocyte activation may participate with microglia in the persistent phase of pain hypersensitivity.     

Phosphoinositide 3-kinase-gamma expression is upregulated in brain microglia and contributes to ischemia-induced microglial activation in acute experimental stroke

Microglia, the resident microphages of the CNS, are rapidly activated after ischemic stroke. Inhibition of microglial activation may protect the brain by attenuating blood-brain barrier damage and neuronal apoptosis after ischemic stroke. However, the mechanisms by which microglia is activated following cerebral ischemia is not well defined. In this study, we investigated the expression of PI3Kγ in normal and ischemic brains and found that PI3Kγ mRNA and protein are constitutively expressed in normal brain microvessels, but significantly upregulated in postischemic brain primarily in activated microglia following cerebral ischemia. In vitro, the expression of PI3Kγ mRNA and protein was verified in mouse brain endothelial and microglial cell lines. Importantly, absence of PI3Kγ blocked the early microglia activation (at 4 h) and subsequent expansion (at 24-72 h) in PI3Kγ knockout mice. The results suggest that PI3Kγ is an ischemia-responsive gene in brain microglia and contributes to ischemia-induced microglial activation and expansion.     

Participation of protease-activated receptor-1 in thrombin-induced microglial activation

Activation of microglia, the resident macrophages in the CNS, plays a significant role in neuronal death or degeneration in a broad spectrum of CNS disorders. Recent studies indicate that nanomolar concentrations of the serine protease, thrombin, can activate microglia in culture. However, in contrast to other neural cells responsive to thrombin, the participation of novel protease-activated receptors (PARs), such as the prototypic thrombin receptor PAR1, in thrombin-induced microglial activation was cast in doubt. In this report, by utilizing primary microglial cultures from PAR1 knockout (PAR1-/-) mice, application of the PAR1 active peptide TRAP-6 (SFLLRN) in comparison to a scrambled peptide (LFLNR), we have unambiguously demonstrated that murine microglia constitutively express PAR1 mRNA that is translated into fully functional protein. Activation of the microglial PAR1 induces a rapid cytosolic free [Ca2+]i increase and transient activation of both p38 and p44/42 mitogen-activated protein kinases. Moreover, although in part, this PAR1 activation directly contributes to thrombin-induced microglial proliferation. Furthermore, although not directly inducing tumor necrosis factor-alpha (TNF-alpha) release, PAR1 activation up-regulates microglial CD40 expression and potentiates CD40 ligand-induced TNF-alpha production, thus indirectly contributing to microglial activation. Taken together, these results demonstrate an essential role of PAR1 in thrombin-induced microglial activation. In addition, strategies aimed at blocking thrombin signaling through PAR1 may be therapeutically valuable for diseases associated with cerebral vascular damage and significant inflammation with microglial activation.     

Tumour necrosis factor alpha-induced neuronal loss is mediated by microglial phagocytosis

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine, expressed in many brain pathologies and associated with neuronal loss. We show here that addition of TNF-α to neuronal–glial co-cultures increases microglial proliferation and phagocytosis, and results in neuronal loss that is prevented by eliminating microglia. Blocking microglial phagocytosis by inhibiting phagocytic vitronectin and P2Y₆ receptors, or genetically removing opsonin MFG-E8, prevented TNF-α induced loss of live neurons. Thus TNF-α appears to induce neuronal loss via microglial activation and phagocytosis of neurons, causing neuronal death by phagoptosis.