Anti-TLR2 antibody triggers oxidative phosphorylation in microglia and increases phagocytosis of β-amyloid

Abstract

BackgroundMicroglia 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.MethodsHere, 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).ResultsWe 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.ConclusionsThus, 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.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1281-7) contains supplementary material, which is available to authorized users.

Iron accumulation in microglia triggers a cascade of events that leads to altered metabolism and compromised function in APP/PS1 mice

“…Here we show that IFNγ+Aβ predictably increased TNFα and iNOS in microglia and that this was paralleled by a switch toward a glycolytic phenotype indicating that glycolysis is increased in inflammatory microglia as it is in inflammatory macrophages. IFNγ alone is also sufficient to induce similar responses in microglia and in BV2 cells but our data indicate that Aβ alone did not significantly increase glycolysis, but in combination with LPS, it induced a significant increase . In the present study, despite the increase in glycolysis, there was no evidence of a change in oxidative metabolism in IFNγ+Aβ‐treated microglia.…”

“…The present data and our previous findings suggest that increased glycolysis in microglia, irrespective of the stimulus, is tightly coupled to increased PFKFB3 . Therefore, as a proxy measure of glycolysis, we assessed PFKFB3 in tissue sections and isolated microglia prepared from APP/PS1 mice, in which IFNγ and Aβ was increased in vivo , compared with WT mice.…”

“…Among the factors that trigger activation of PFKFB3 are inflammatory mediators; for example, IL‐6 increased PFKFB3 mRNA in mouse embryonic fibroblasts and cancer cell lines and several studies have indicated that LPS triggers PFKFB3 in macrophages . Few studies have investigated factors that drive PFKFB3 in microglia but we have reported that IFNγ alone and also LPS+Aβ exert this effect. HIF‐1α is recognized as a potent activator of PFKFB3 and this has been described in fibroblasts and macrophages .…”

“…Briefly, isolated mixed glia from the cortical tissue of C57BL/6 neonatal mice were cultured in T25 cm 2 flasks in cDMEM supplemented with macrophage colony stimulating factor (M‐CSF; 100 ng/mL; R&D Systems, Abingdon, UK) and granulocyte macrophage colony stimulating factor (GM‐CSF; 100 ng/mL; R&D Systems, Abingdon, UK) for 10–12 days, after which time non‐adherent microglia were seeded in 24‐well plates (1 × 10 5 cells/well) and cultured for a further 2 days. Medium was replaced with fresh cDMEM ± IFNγ (50 ng/mL; Enzo Life Sciences, Exeter, UK) and amyloid‐β [Aβ; 10 μM comprising Aβ 1‐40 (4.2 μM) + Aβ 1‐42 (5.8 μM)] for 24 h. We used this Aβ combination because cells are exposed to both peptides in vivo and previous experiments indicated that it triggered inflammatory changes and synergized with LPS to trigger neuroinflammatory and metabolic changes . To prepare Aβ, lyophilized Aβ 1‐40 and Aβ 1‐42 peptides were dissolved in HPLC grade water to provide a 6 mg/mL stock solution, which was diluted to 1 mg/mL using sterile PBS and allowed to aggregate (24 h, 220 rpm, 37°C).…”

Metabolic Reprograming of Microglia in the Regulation of the Innate Inflammatory Response

“…On the other hand, primary rat microglia cultured with increasing glucose concentration (from 10 to 50 mM) boosted TNFα secretion (58,59). More recently, Rubio-Araiz et al showed that primary microglia exposure to LPS and amyloid-β (Aβ) induced an inflammatory state associated with the increase of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), with a boost in extracellular acidification rate (ECAR) (60). IFNγ and Aβ also increased microglia glycolysis together with an increase in PFKFB3, hexokinase II and Pyruvate kinase isozymes M2 (PKM2) (61), suggesting that inflammation affects microglia metabolism, driving the glycolysis pathway through increased PFKFB3 activation.…”