An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer’s disease) to development of the healthy brain (e.g., synaptic pruning)^1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system⁷. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).     

Long-range Ca2+ waves transmit brain-damage signals to microglia

Microglia are the resident phagocytes of the brain that are responsible for the clearance of injured neurons, an essential step in subsequent tissue regeneration. How death signals are controlled both in space and time to attract these cells toward the site of injury is a topic of great interest. To this aim, we have used the optically transparent zebrafish larval brain and identified rapidly propagating Ca2+ waves that determine the range of microglial responses to neuronal cell death. We show that while Ca2+-mediated microglial responses require ATP, the spreading of intercellular Ca2+ waves is ATP independent. Finally, we identify glutamate as a potent inducer of Ca2+-transmitted microglial attraction. Thus, this real-time analysis reveals the existence of a mechanism controlling microglial targeted migration to neuronal injuries that is initiated by glutamate and proceeds across the brain in the form of a Ca2+ wave.     

From regulation of dying cell engulfment to development of anti-cancer therapy

Cell death and efficient engulfment of dying cells ensure tissue homeostasis and is involved in pathogenesis. Clearance of dying cells is a complex and dynamic process coordinated by interplay between ligands on dying cell, bridging molecules, and receptors on engulfing cells. In this review, we will discuss recent advances and significance of molecular changes on the surface of dying cells implicated in their recognition and clearance as well as factors released by dying cells that attract macrophages to the site of cell death. It is now becoming apparent that phagocytes use a specific set of mechanisms to discriminate between live and dead cells, and this phenomenon will be illustrated here. Next, we will discuss potential mechanisms by which removal of dying cells could modulate immune responses of phagocytes, in particular of macrophages. Finally, we will address possible strategies for manipulating the immunogenicity of dying cells in experimental cancer therapies.     

Cholesterol-induced apoptotic macrophages elicit an inflammatory response in phagocytes, which is partially attenuated by the Mer receptor

Macrophage apoptosis and the ability of phagocytes to clear these apoptotic cells are important processes in advanced atherosclerosis. Phagocytic clearance not only disposes of dead cells but usually elicits an anti-inflammatory response. To study this process in a model of advanced lesional macrophage death, macrophages rendered apoptotic by free cholesterol loading (FC-AMs) were incubated briefly with fresh macrophages ("phagocytes"). FC-AMs were promptly ingested by the phagocytes, which was dependent upon actin polymerization and the phagocyte Mer receptor. Surprisingly, this brief exposure to FC-AMs triggered a modest proinflammatory response in the phagocytes: tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta were induced, whereas the levels of transforming growth factor-beta and IL-10 were not increased. This response required cell contact between the FC-AMs and phagocytes but not FC-AM ingestion. TNF-alpha and IL-1beta induction required one or more proteins on the FC-AM surface and was dependent on signaling through extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase and nuclear factor-kappaB in the phagocytes. TNF-alpha production was markedly greater when Mer-defective phagocytes were used, indicating that Mer attenuated the inflammatory response. Interestingly, a more typical anti-inflammatory response was elicited when phagocytes were exposed to macrophages rendered apoptotic by oxidized low density lipoprotein or UV radiation. Thus, the proinflammatory milieu of advanced atherosclerotic lesions may be promoted, or at least not dampened, by contact between FC-induced apoptotic macrophages and neighboring phagocytes prior to apoptotic cell ingestion.     

Microglia: phagocyte and glia cell

Microglia are the resident immune cells of the brain, and are located within the brain parenchyme behind the blood-brain barrier. They originate from mesodermal hemapoietic precursors and are slowly turned over and replenished by proliferation in the adult central nervous system. In the healthy brain resting, ramified microglia function as supportive glia cells, and their activation status is regulated by neurons through soluble mediators and cell-cell contact. However, in response to brain pathology microglia become activated: acquisition of innate immune cell functions render microglia competent to react towards brain injury through tissue repair or induction of immune responses. In certain pathological conditions, however, microglia activation may sustain a chronic inflammation of the brain, leading to neuronal dysfunction and cell death. This might be mediated by the microglial release of extracellular toxic reactive oxygen and nitrogen species. Nevertheless, in the future microglia may potentially be harnessed for therapeutical purposes.     

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.     

Microglial phagocytosis of apoptotic inflammatory T cells leads to down-regulation of microglial immune activation.

Apoptotic cell death is an established mechanism to terminate an inflammatory response in rodent or human brains. Microglia, as the resident phagocyte, is a strong candidate for the clearance of apoptotic lymphocytes. Apoptosis was induced in cultured autologous thymocytes and in myelin basic protein (MBP)-specific, encephalitogenic T cells from Lewis rats by the addition of 0.1 microg/ml methylprednisolone. The amount of phagocytosis of apoptotic cells was assessed using an in vitro phagocytosis assay. Supernatants were collected to measure microglial cytokine secretion. The state of immune activation in microglia was investigated by a T cell proliferation assay and by flow cytometric analysis of microglial surface expression of immune molecules. Microglia ingested specifically apoptotic cells (apoptotic thymocytes as well as MBP-specific T cells) in contrast to nonapoptotic control cells (p < 0.0001). Subsequent secretion of the proinflammatory cytokines TNF-alpha and IL-12 was significantly decreased, while the secretion of IL-10 and TGF-beta was not affected. Furthermore, ingestion of apoptotic cells led to increased microglial MHC class II expression without concomitant increase in MHC class I, costimulatory molecules, and ICAM expression. The Ag-specific activation of MBP-specific T cells in cocultures with microglia that had ingested apoptotic cells was significantly less than that of identical T cells that interacted with nonphagocytosing microglia. Together with negative results obtained in a trans-well system, this is in support of a cell contact-mediated effect. Microglia might play an important role in the clearance of apoptotic cells. The uptake of apoptotic cells by microglia is tolerogenic and results in a reduced proinflammatory cytokine production and a reduced activation of encephalitogenic T cells. This might help to restrict an autoimmune inflammation and minimize damage in the inflamed brain.     

Development of ramified microglia from early macrophages in the zebrafish optic tectum

Microglia, the resident macrophage precursors of the brain, are necessary for the maintenance of tissue homeostasis and activated by a wide range of pathological stimuli. They have a key role in immune and inflammatory responses. Early microglia stem from primitive macrophages, however the transition from early motile forms to the ramified mature resident microglia has not been assayed in real time. In order to provide such an assay, we used zebrafish transgenic lines in which fluorescent reporter expression is driven by the promoter of 1 (mpeg1; Ellet et al. [2011]: Blood 117(4): e49–e56,). This enabled the investigation of the development of these cells in live, intact larvae. We show that microglia develop from highly motile amoeboid cells that are engaged in phagocytosis of apoptotic cell bodies into a microglial cell type that rapidly morphs back and forth between amoeboid and ramified morphologies. These morphing microglia eventually settle into a typical mature ramified morphology. Developing microglia frequently come into contact with blood capillaries in the brain, and also frequently contact each other. Up to 10 days postfertilization, microglia were observed to undergo symmetric division. In the adult optic tectum, the microglia are highly branched, resembling mammalian microglia. In addition, the mpeg1 transgene also labeled highly branched cells in the skin overlying the optic tectum from 8–9 days postfertilization, which likely represent Langerhans cells. Thus, the development of zebrafish microglia and their cellular interactions was studied in the intact developing brain in real time and at cellular resolution. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

The disposal of dying cells in living tissues

Cells continuously die and disappear from the midst of living tissues. However, some of their constituents survive. DNA is horizontally transferred to phagocytic cells, and apoptotic cell antigens shape the immune repertoire. When massive apoptosis occurs, which overwhelms tissue scavenger cells, or when the function of phagocytes abates, dying cells escape clearance in vivo. Remnant dying cells come to phagocytes disguised: factors capable to envelop their membranes pervade the entire organism, or are generated in given tissues. Some are constitutively present, while other are generated during early or late phases of the inflammatory response, possibly to face the further burden of the dead inflammatory cells. This camouflage influences the disposal of the corpses: decoying molecules either bridge the corpse to the phagocyte or hide it. Furthermore, factors associated to the plasma membrane of the apoptotic cell shape the signals the phagocyte releases in situ. Finally, molecules contained or released by the dying cell alter the apprehension by the phagocyte of its prey, influencing its immunogenicity.     

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare ''professional'' phagocytes - macrophages and dendritic cells to ''non-professional'' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: ''professional'' macrophages and ''semi-professional'' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.     

Temporary colonization of the site of lesion by macrophages is a prelude to the arrival of regenerated axons in injured goldfish optic nerve

Summary. In crushed goldfish optic nerve, regenerating axons cross the site of lesion within 10 days following injury. Some 30 days later, Schwann cells accumulate at the lesion, where they myelinate the new axons. In this study, we have used immunohistochemistry and electron microscopy to examine the cellular environment of the crush site prior to the establishment of Schwann cells in order to learn more about the early events that contribute to axonal regeneration. During the first week following injury, macrophages enter the site of lesion and efficiently phagocytose the debris. The infiltration of macrophages precedes the arrival of regenerating axons that abut and surround these phagocytes. Based on EM morphology and phagocytic capacity, macrophages of the type observed at the site of lesion are not present in the degenerating distal nerve segment, where debris clearance is shared between conventional microglia and astrocytes over a period of several weeks. During this period, axon bundles emerging distally from the injury zone become enwrapped by astrocyte processes, thereby re-establishing the characteristic fascicular cytoarchitecture of the optic nerve. The process of fasciculation also leads to the displacement of myelin debris to the margins of the fiber bundles, where it is trapped by the astrocytes. Our results suggest that the early robust appearance of macrophages at the lesion, and their effectiveness as phagocytes compared with the microglia distally, may contribute to the vigorous axonal regeneration across the crush, beyond which axons<197>excepting the pioneers<197>extend through newly formed debris-free channels delineated by astrocyte processes.     

Yolk sac-derived Pdcd11-positive cells modulate zebrafish microglia differentiation through the NF-κB-Tgfβ1 pathway

Microglia are the primary immune cells in the central nervous system, which plays a vital role in neuron development and neurodegenerative diseases. Microglial precursors in peripheral hematopoietic tissues colonize the central nervous system during early embryogenesis. However, how intrinsic and extrinsic signals integrate to regulate microglia’s differentiation remains undefined. In this study, we identified the cerebral white matter hyperintensities susceptibility gene, programmed cell death protein 11 (PDCD11), as an essential factor regulating microglia differentiation. In zebrafish, pdcd11 deficiency prevents the differentiation of the precursors to mature brain microglia. Although, the inflammatory featured macrophage brain colonization is augmented. At 22 h post fertilization, the Pdcd11-positive cells on the yolk sac are distinct from macrophages and neutrophils. Mechanistically, PDCD11 exerts its physiological role by differentially regulating the functions of nuclear factor-kappa B family members, P65 and c-Rel, suppressing P65-mediated expression of inflammatory cytokines, such as tnfα, and enhancing the c-Rel-dependent appearance of tgfβ1. The present study provides novel insights in understanding microglia differentiation during zebrafish development.Subject terms: Cell biology, Molecular biology     

The immunomodulatory sphingosine 1-phosphate analog FTY720 reduces lesion size and improves neurological outcome in a mouse model of cerebral ischemia

Cerebral ischemia is accompanied by fulminant cellular and humoral inflammatory changes in the brain which contribute to lesion development after stroke. A tight interplay between the brain and the peripheral immune system leads to a biphasic immune response to stroke consisting of an early activation of peripheral immune cells with massive production of proinflammatory cytokines followed by a systemic immunosuppression within days of cerebral ischemia that is characterized by massive immune cell loss in spleen and thymus. Recent work has documented the importance of T lymphocytes in the early exacerbation of ischemic injury. The lipid signaling mediator sphingosine 1-phosphate-derived stable analog FTY720 (fingolimod) acts as an immunosuppressant and induces lymphopenia by preventing the egress of lymphocytes, especially T cells, from lymph nodes. We found that treatment with FTY720 (1 mg/kg) reduced lesion size and improved neurological function after experimental stroke in mice, decreased the numbers of infiltrating neutrophils, activated microglia/macrophages in the ischemic lesion and reduced immunohistochemical features of apoptotic cell death in the lesion.     

Engulfing astrocytes protect neurons from contact-induced apoptosis following injury

Clearing of dead cells is a fundamental process to limit tissue damage following brain injury. Engulfment has classically been believed to be performed by professional phagocytes, but recent data show that non-professional phagocytes are highly involved in the removal of cell corpses in various situations. The role of astrocytes in cell clearance following trauma has however not been studied in detail. We have found that astrocytes actively collect and engulf whole dead cells in an in vitro model of brain injury and thereby protect healthy neurons from bystander cell death. Time-lapse experiments showed that migrating neurons that come in contact with free-floating cell corpses induced apoptosis, while neurons that migrate through groups of dead cells, garnered by astrocytes, remain unaffected. Furthermore, apoptotic cells are present within astrocytes in the mouse brain following traumatic brain injury (TBI), indicating a possible role for astrocytes in engulfment of apoptotic cells in vivo. qRT-PCR analysis showed that members of both ced pathways and Megf8 are expressed in the cell culture, indicating their possible involvement in astrocytic engulfment. Moreover, addition of dead cells had a positive effect on the protein expression of MEGF10, an ortholog to CED1, known to initiate phagocytosis by binding to phosphatidylserine. Although cultured astrocytes have an immense capacity for engulfment, seemingly without adverse effects, the ingested material is stored rather than degraded. This finding might explain the multinuclear astrocytes that are found at the lesion site in patients with various brain disorders.     

PK (‘peripheral benzodiazepine’) – binding sites in the CNS indicate early and discrete brain lesions: microautoradiographic detection of [3H]PK 11195 binding to activated microglia

The isoquinoline PK 11195 has been suggested as a marker of glial pathology in the lesioned brain. The aim of the present study is to clarify the precise cellular location of its binding site in the central nervous system. Here, we report that in the facial nucleus after facial nerve axotomy–a lesion causing a retrograde neuronal reaction without nerve cell death while keeping the blood–brain barrier intact–activated microglia are the predominant source of lesion-induced increases of PK 11195 binding. Likewise, increased PK 11195 binding is seen in the gracile nucleus after anterograde neuronal injury following sciatic nerve transection. The peak of PK 11195 binding, using the single isomer R-PK 11195, was observed 4 days after the peripheral nerve lesion, consistent with the well-known time course of microglial activation. Photoemulsion microautoradiography confirmed the restriction of PK 11195 binding to activated microglia. The increase of PK 11195 binding in the facial nucleus seen after selective cell death of facial motoneurons by retrograde suicide transport of toxic ricin, a lesion that is accompanied by the rapid transformation of microglia into phagocytes, was no higher than that seen following axotomy. This suggests that the full transformation of microglia into parenchymal phagocytes is not necessary to reach maximal levels of PK 11195 binding. PK 11195, therefore, is a well-suited marker to detect microglial activation in areas of subtle brain pathology, where neither a disturbance of the blood–brain barrier function nor the presence of macrophages and inflammatory cells indicate an on-going disease process.     

Enhanced microglial pro‐inflammatory response to lipopolysaccharide correlates with brain infiltration and blood–brain barrier dysregulation in a mouse model of telomere shortening

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self‐renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as ‘priming’. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first‐generation G1 mTerc^?/?)‐ and late‐generation (third‐generation G3 and G4 mTerc^?/?) telomerase‐deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late‐generation mTerc^?/? microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc^?/? microglia are comparable with microglia derived from G1 mTerc^?/? mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc^?/? microglia mice show an enhanced pro‐inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age‐associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood–brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.     

Microglia: activation and their significance in the central nervous system

Microglia are resident monocyte-lineaged cells in the brain. Their characteristic feature is that they react to injury and diseases of the brain and become morphologically and functionally activated. Although some trigger molecules which activate microglia are predicted to be released from injured or affected cells, such molecules have not yet been identified. The main role of activated microglia is believed to be in brain defense, as scavengers of dead cells, and as immune or immunoeffector cells. Recent biochemical and neurobiological studies have further indicated that they significantly affect the pathological state and/or regulate the regenerative state and remodeling of the brain by producing a variety of biologically active molecules including cytotoxic and neurotrophic molecules.     

Invasion of microglia/macrophages and granulocytes into the Mauthner axon myelin sheath following spinal cord injury of the adult goldfish,Carassius auratus

Rapid activation of resident glia occurs after spinal cord injury. Somewhat later, innate and adaptive immune responses occur with the invasion of peripheral immune cells into the wound site. The activation of resident and peripheral immune cells has been postulated to play harmful as well as beneficial roles in the regenerative process. Mauthner cells, large identifiable neurons located in the hindbrain of most fish and amphibians, provided the opportunity to study the morphological relationship between reactive cells and Mauthner axons (M-axons) severed by spinal cord crush or by selective axotomy. After crossing in the hindbrain, the M-axons of adult goldfish, Carassius auratus, extend the length of the spinal cord. Following injury, the M-axon undergoes retrograde degeneration within its myelin sheath creating an axon-free zone (proximal dieback zone). Reactive cells invade the wound site, enter the axon-free dieback zone and are observed in the vicinity of the retracted M-axon tip as early as 3 hr postinjury. Transmission electron microscopy allowed the detection of microglia/macrophages and granulocytes, some of which appear to be neutrophil-like, at each of these locations. We believe that this is the first report of the invasion of such cells within the myelin sheath of an identifiable axon in the vertebrate central nervous system (CNS). We speculate that microglia/macrophages and granulocytes that are attracted within a few hours to the damaged M-axon are part of an inflammatory response that allows phagocytosis of debris and plays a role in the regenerative process. Our results provide the baseline from which to utilize immunohistochemical and genetic approaches to elucidate the role of non-neuronal cells in the regenerative process of a single axon in the vertebrate CNS.     

Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa , suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.