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Inhibition of Microglial Phagocytosis Is Sufficient To Prevent Inflammatory Neuronal Death

Jonas J. Neher, Urte Neniskyte, Jing-Wei Zhao, Anna Bal-Price, Aviva M. Tolkovsky and Guy C. Brown
J Immunol April 15, 2011, 186 (8) 4973-4983; DOI: https://doi.org/10.4049/jimmunol.1003600
Jonas J. Neher
*Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom;
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Urte Neniskyte
*Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom;
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Jing-Wei Zhao
†Cambridge Centre for Brain Repair, University of Cambridge, Cambridge CB2 OPY, United Kingdom; and
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Anna Bal-Price
‡In Vitro Toxicology Unit, European Centre for the Validation of Alternative Methods, Institute of Health and Consumer Protection, European Commission Joint Research Centre, 21027 Ispra, Italy
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Aviva M. Tolkovsky
*Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom;
†Cambridge Centre for Brain Repair, University of Cambridge, Cambridge CB2 OPY, United Kingdom; and
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Guy C. Brown
*Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom;
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  • FIGURE 1.
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    FIGURE 1.

    Inflammatory loss of neurons is mediated by microglial phagocytosis. A, LTA (50 μg/ml) activation of mixed cultures for 3 d results in neuronal loss without death (left panel) and proliferation of microglia (right panel). Elimination of microglia with LME prevents neuronal loss (necrotic cells also include lysed microglia in LME-treated cultures). B, Left panel, Activation of microglia with LTA (50 μg/ml), LPS (100 ng/ml), or GM-CSF (50 ng/ml) for 24 h strongly increases phagocytic uptake of carboxylate-modified microspheres (1 μm), mimicking the negatively charged surface of PS-exposing cells. Right panel, Fluorescence image of microbead uptake (orange) by nonactivated and LTA-activated microglia stained with isolectin-B4 (green) and Hoechst 33342 (nuclei, blue). C, GM-CSF (50 ng/ml) stimulates microglial proliferation (right panel) and phagocytic activity (see B) but does not cause neuronal loss over 7 d (left panel). D, Single images from time-lapse video analysis of a mixed culture treated with LTA (frame rate, 0.5 h). Yellow and green arrows identify two neurons that are phagocytosed by a microglial cell (red arrows). Note the absence of neuronal morphological changes before engulfment. Data shown are means ± SEM for three or more independent experiments; **p < 0.01, ***p < 0.001 versus control, ###p < 0.001 versus LTA.

  • FIGURE 2.
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    FIGURE 2.

    Neuronal loss requires release of soluble inflammatory mediators and direct neuronal–microglial contact. A, LTA-induced neuronal loss over 3 d is prevented by separation of neurons/astrocytes from microglia by TWs, without increasing the densities of dying cells (necrotic cells not shown, because LME treatment leads to microglial lysis and chromatin release; cf. Fig. 1). ***p < 0.001 versus control, ###p < 0.001 versus LTA. B, Microglial densities for experiment in A. *p < 0.05, **p < 0.01 versus respective controls. C, Separation of inflammatory soluble mediator release and direct microglial–neuronal contact using transwell cocultures (TW). First column from left, Microglia-depleted cultures were left untreated; second column, cultures were stimulated with LTA for 2 d, and LTA-activated microglia were then added for 6 h; third column, microglia-depleted cultures were exposed to nonactivated microglia on transwells for 2 d followed by addition of LTA-activated microglia for 6 h; fourth column, cultures were exposed to LTA-activated microglia on transwells for 2 d followed by LTA-activated microglia for 6 h. Delayed addition of LTA-activated microglia causes neuronal loss only if neurons were pre-exposed to soluble inflammatory mediators from LTA-activated transwell microglia. ##p < 0.01 versus TW−. Data shown are means ± SEM for three or more independent experiments.

  • FIGURE 3.
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    FIGURE 3.

    Inflammatory neuronal eat-me signaling is reversible and mediated by RONS. A, Removal of transwell microglia for 1 d after 3 d of LTA (50 μg/ml) stimulation leads to decreased neuronal loss on addition of activated microglia (cf. Fig. 2), indicating reversibility of neuronal eat-me signaling. ***p < 0.001 versus TW−, ##p < 0.01 for TW LTA versus TW LTA − TW removed). B, Confocal micrographs of neurons below transwells (astrocytes lie in a layer underneath the focal plane) loaded with fluorescent PS (NBD-PS, green) at 3 d after LTA stimulation. Nuclei are counterstained with propidium iodide (red) after fixation. Note the reduced signal intensity of LTA-stimulated cultures (TW LTA) compared with control cultures (TW control) after quenching of exoplasmic NBD-PS, indicating increased PS exposure. Scale bars, 50 μm. C, LTA stimulation of transwell cultures for 3 or 4 d increases PS exposure (quantified as a decrease in neuronal NBD-PS fluorescence after quenching of exposed NBD-PS). Blocking endocytosis with cytochalasin D (Cyto D) has no effect on NBD-PS levels. The LTA-induced PS exposure is reversed by removing the transwell microglia or by scavenging superoxide (using SOD) or peroxynitrite (using FeTPPS) for 1 d after 3 d of LTA stimulation. For all bars, the NBD-PS fluorescence of quenched neurons is expressed as percentage of NBD-PS signal for untreated transwell cultures. D, LTA- (50 μg/ml) or LPS-induced (100 ng/ml) neuronal loss is prevented by delayed scavenging (added 2 d after LTA/LPS) of RONS using SOD, the SOD mimetic MnTBAP, or the peroxynitrite scavenger FeTPPS, or by inhibition of NO synthases using L-NAME, for 1 d. In contrast, catalase (CAT) has no effect. Neuronal density and death was quantified 3 d after LTA or LPS addition to mixed cultures. Microglial activation/proliferation was unaffected by all treatments (see Supplemental Table I). Data shown are means ± SEM for three or more independent experiments. **p < 0.01, ***p < 0.001 versus control; #p < 0.05, ##p < 0.01, ###p < 0.001 versus LTA or LPS.

  • FIGURE 4.
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    FIGURE 4.

    Nontoxic levels of peroxynitrite cause reversible PS exposure and microglia-dependent neuronal loss. A, Low levels of peroxynitrite (10 μM) cause loss of neurons over 3 d without accumulation of dying cells in mixed cultures. B, In mixed cultures, peroxynitrite increases neuronal PS exposure (Annexin V+ cells) and chromatin-condensation between 1 h and 1 d after treatment, returning to baseline at 2 d, concomitant with a decrease in healthy cells (0 h indicates staining immediately after addition of peroxynitrite). C, Peroxynitrite (10 μM) does not cause significant neuronal death or loss in microglia-depleted cultures even 7 d after peroxynitrite addition (cf. A). D, In microglia-depleted cultures, peroxynitrite (10 μM) induces reversible chromatin condensation and PS exposure, but no loss of neurons. Data shown are means ± SEM for three or more independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

  • FIGURE 5.
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    FIGURE 5.

    Inhibition of phagocytosis prevents LTA-, LPS-, peroxynitrite- and Aβ-induced neurodegeneration, leaving behind healthy neurons. A, Neuronal loss induced by LTA (50 μg/ml) or LPS (100 ng/ml) is reduced by inhibiting the PS–MFG-E8–vitronectin receptor system. Significant neuroprotection is provided by: 1) preventing PS recognition through addition of C2A domain, 2) blocking MFG-E8 bridging of PS-exposing cells and the vitronectin receptor on microglia using a MFG-E8 blocking Ab, or 3) using the vitronectin receptor-specific blocking peptide cRGD to prevent MFG-E8 recognition. In contrast, a control IgG and cRAD have no effect. B, Protection provided by cRGD (50 μM) against LPS (100 ng/ml)-induced neuronal loss does not lead to accumulation of caspase-3+ cells 3 d after stimulation. C, Loading with TMRM (3 nM) indicates that the plasmalemmal and the mitochondrial membrane potential are intact in neurons protected from LPS (100 ng/ml)-induced neuronal loss by cRGD (50 μM). Dark bars represent the number of healthy neurons as a percentage of that in the untreated control culture; light bars represent the total number of neurons positive for TMRM as percentage of that in the untreated control culture. Thus, in each treatment, virtually all neurons present are TMRM+. D, Treatment of mixed cultures with nontoxic levels of peroxynitrite and SIN-1 leads to significant neuronal loss after 3 d. This does not occur after decomposition of either reagent in culture medium (“decomposed”). The SIN-1 effect is mediated by peroxynitrite as shown by prevention of neuronal loss with the peroxynitrite scavenger FeTPPS. The vitronectin receptor antagonist, cRGD, protects against neuronal loss induced by low peroxynitrite (5 μM), and partially protects against intermediate peroxynitrite (10 μM) and SIN-1 (50 μM), but not high peroxynitrite (20 μM), which induces significant cell death by necrosis and apoptosis. E, Treatment of mixed cultures with the β-amyloid peptide (25–35) (Aβ[25–35], 250 nM) leads to significant neuronal loss over 3 d, whereas the reverse peptide Aβ(35–25) has no effect on neuronal density. Aβ(25–35)-induced neuronal loss is not prevented by incubation with polymyxin B (PMXN B), excluding LPS contamination. In contrast, elimination of microglia using LME treatment, delayed addition of the peroxynitrite scavenger FeTPPS (2 d after stimulation), or cotreatment with the vitronectin receptor antagonist, cRGD, significantly prevents Aβ(25–35) induced neuronal loss. The control peptide cRAD, in contrast, has no effect. Gray lines indicate 100% and the approximate level of neuronal loss for ease of comparison. Data shown are means ± SEM for three or more independent experiments. **p < 0.01, ***p < 0.001 compared with control; #p < 0.05, ##p < 0.01 compared with LTA/LPS/peroxynitrite/SIN-1/Aβ(25–35).

  • FIGURE 6.
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    FIGURE 6.

    MFG-E8 is located on microglia and neurons, and acts as a bridging molecule for phagocytic recognition. A–O, Differential interference contrast (DIC, A–C) and confocal fluorescent images showing live staining of mixed cultures for MFG-E8 (red) and microglia (isolectin-B4, green) 3 d after treatment. As reported for macrophages, MFG-E8 appears to be localized in vesicles on the microglial surface both in control and LPS (100 ng/ml)-activated cultures (white arrows). MFG-E8 opsonized cells (yellow arrows) are only rarely found in control cultures (D) and are absent in LPS-treated cultures (E), consistent with their phagocytic removal. In contrast, cells positive for MFG-E8 can be observed in cultures treated with LPS in combination with vitronectin receptor antagonist cRGD (cRGDfV, 50 μM) (F), indicating inhibition of phagocytosis of MFG-E8–opsonized neurons. Scale bars, 10 μm. The scheme shows the proposed mechanism of primary phagocytosis in inflammatory neurodegeneration.

Additional Files

  • Figures
  • Data Supplement

    Files in this Data Supplement:

    • Supplemental Table I, Figures 1-5 and Video Legend (PDF, 347 Kb) - Description:
      Table I. The table presents indicators of inflammation for experiments described in the main text...
      Figure 1. Astrocytes are not affected by treatment with L-leucinemethyl-ester...
      Figure 2. Inflammatory activation of mixed cultures with LTA (50 μg/ml) leads to microglial superoxide production...
      Figure 3. The integrin-receptor antagonist, RGDS, reduces LTAinduced neuronal loss...
      Figure 4. cycloRGDfV has no direct neuroprotective effect...
      Figure 5. Hydrogen peroxide induces reversible phosphatidylserineexposure in the absence of neuronal death...
    • Video 1 (MOV, 599 Kb) - Neuronal loss is mediated by microglial phagocytosis of apparently healthy neurons...
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The Journal of Immunology: 186 (8)
The Journal of Immunology
Vol. 186, Issue 8
15 Apr 2011
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Inhibition of Microglial Phagocytosis Is Sufficient To Prevent Inflammatory Neuronal Death
Jonas J. Neher, Urte Neniskyte, Jing-Wei Zhao, Anna Bal-Price, Aviva M. Tolkovsky, Guy C. Brown
The Journal of Immunology April 15, 2011, 186 (8) 4973-4983; DOI: 10.4049/jimmunol.1003600

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Inhibition of Microglial Phagocytosis Is Sufficient To Prevent Inflammatory Neuronal Death
Jonas J. Neher, Urte Neniskyte, Jing-Wei Zhao, Anna Bal-Price, Aviva M. Tolkovsky, Guy C. Brown
The Journal of Immunology April 15, 2011, 186 (8) 4973-4983; DOI: 10.4049/jimmunol.1003600
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