The online version of this article (https://doi.org/10.1186/s12974-018-1281-7) contains supplementary material, which is available to authorized users.
Microglia are multifunctional cells that are primarily neuroprotective and a deficit in their functional integrity is likely to be a contributory factor in the deteriorating neuronal function that occurs with age and neurodegeneration. One aspect of microglial dysfunction is reduced phagocytosis, and this is believed to contribute to the accumulation of amyloid-β (Aβ) in Alzheimer’s disease (AD). Therefore, improving phagocytosis should be beneficial in limiting the amyloidosis that characterises AD.
Here, we investigated whether an antibody that targets toll-like receptor (TLR)2 might attenuate the inflammatory and metabolic changes induced by lipopolysaccharide (LPS) and amyloid-β. The impact on phagocytosis was assessed by immunohistochemistry. We evaluated the metabolic changes with the SeaHorse Extracellular Flux Analyser and studied the expression of key enzymes driving glycolysis by western blotting. For all experiments, statistical significance was determined by unpaired Student’s t test and two-way analysis of variance (ANOVA).
We have reported that, when exposed to an inflammatory stimulus, microglia switch their metabolism towards the metabolically- inefficient glycolysis; this potentially impacts on metabolically demanding functions like phagocytosis. Anti-TLR2 antibody increased phagocytosis of Aβ in LPS + Aβ-stimulated microglia and this was linked with the ability of the antibody to attenuate the LPS + Aβ-triggered inflammasome activation. LPS + Aβ increased glycolysis in microglia and increased the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3, an enzyme that plays a key role in driving glycolysis; these effects were inhibited when cells were incubated with the anti-TLR2 antibody. The data also show that antibody treatment increased oxidative metabolism.
Thus, microglia with an inflammatory phenotype, specifically cells in which the inflammasome is activated, are glycolytic; this may compromise the metabolic efficiency of microglia and thereby provide an explanation for the reduced phagocytic function of the cells. We propose that, by restoring oxidative metabolism and reducing inflammasome activation in microglia, phagocytic function is also restored.
Additional file 1: Figure S2. Expression of TLR2 in microglia. Microglia were prepared and treated as described in the legend for Fig. 1 and stained for TLR2 incubating with the primary antibody anti-TLR2 (Abcam, UK) followed by the secondary antibody Alexa Fluor® 488 donkey anti-mouse IgG (1:1000) and mounted in ProLong®Gold with the nuclear marker DAPI (Thermo Scientific, USA). The panel shows fluorescence images at × 40 magnification and shows that TLR2 staining is confined to the membrane of the microglial cells independently of the experimental groups observed. (Scale bar = 50 μm). (PNG 694 kb)
Additional file 2: Figure S1. Anti-TLR2 antibody attenuates the LPS + Aβ-induced glycolytic capacity of microglia but has no effect of LPS-induced or Aβ-induced changes. Microglia were assessed for their metabolic profile using SeaHorse technology following incubation with LPS, Aβ or both in the presence or absence of anti-TLR2 antibody as described in the “Methods” section. LPS + Aβ significantly increased mean glycolytic capacity and glycolysis (*p < 0.05). The LPS + Aβ-induced effect on glycolytic capacity was significantly attenuated when cells were also incubated with anti-TLR2 antibody (+p < 0.05; LPS + Aβ vs LPS + Aβ+anti-TLR2 antibody). The modulatory effect of the anti-TLR2 antibody on LPS + Aβ-induced glycolysis did not reach statistical significance. (PNG 67 kb)
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- Anti-TLR2 antibody triggers oxidative phosphorylation in microglia and increases phagocytosis of β-amyloid
Orla M. Finucane
Marina A. Lynch
- BioMed Central