Abstract
Metabolic regulation of immune cells has arisen as a critical set of processes required for appropriate response to immunological signals. While our knowledge in this area has rapidly expanded in leukocytes, much less is known about the metabolic regulation of brain-resident microglia. In particular, the role of alternative nutrients to glucose remains poorly understood. Here, we use stable-isotope (13C) tracing strategies and metabolomics to characterize the oxidative metabolism of β-hydroxybutyrate (BHB) in human (HMC3) and murine (BV2) microglia cells and the interplay with glucose in resting and LPS-activated BV2 cells. We found that BHB is imported and oxidised in the TCA cycle in both cell lines with a subsequent increase in the cytosolic NADH:NAD+ ratio. In BV2 cells, stimulation with LPS upregulated the glycolytic flux, increased the cytosolic NADH:NAD+ ratio and promoted the accumulation of the glycolytic intermediate dihydroxyacetone phosphate (DHAP). The addition of BHB enhanced LPS-induced accumulation of DHAP and promoted glucose-derived lactate export. BHB also synergistically increased LPS-induced accumulation of succinate and other key immunometabolites, such as α-ketoglutarate and fumarate generated by the TCA cycle. Finally, BHB upregulated the expression of a key pro-inflammatory (M1 polarisation) marker gene, NOS2, in BV2 cells activated with LPS. In conclusion, we identify BHB as a potentially immunomodulatory metabolic substrate for microglia that promotes metabolic reprogramming during pro-inflammatory response.
Highlights
Microglia are the resident immune cells of the central nervous system (CNS) and represent approximately 10% of CNS cells in a healthy brain and spinal cord
body β-hydroxybutyrate (BHB) is oxidised through the three-step ketone body oxidation pathway with the subsequent production of NADH and acetyl-CoA, which can be incorporated into the TCA cycle (Figure 1A)
Using stable-isotope tracing with 13 C-BHB, we have shown that microglia cells can import and oxidise BHB in the TCA cycle with a subsequent increase in the cytosolic NADH:NAD+ ratio
Summary
Microglia are the resident immune cells of the central nervous system (CNS) and represent approximately 10% of CNS cells in a healthy brain and spinal cord. In brain disease or upon immune challenge, resting microglia adopt programmatic changes associated with the release of cytokines and chemokines. These polarised cells have been traditionally categorised as having either a pro-inflammatory (M1 type) or anti-inflammatory (M2 type) states depending on the expression of a few molecular markers. Observations in immunometabolism have reported that peripheral immune cells can adapt to fluctuating environmental challenges by metabolising alternative nutrients other than glucose, such as acetate [14], amino acids [15], or fatty acids [13,16] In microglia, this phenomenon of the so-called metabolic flexibility and the utilisation of alternative substrates other that glucose is still poorly understood. We assessed the effect of BHB on the inflammatory response to LPS by analysing changes in the expression of polarisation makers
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