| dc.description.abstract | Immunometabolism is the study of changes that occur in the metabolic pathways of an immune cell during its activation. For macrophages, the remodelling of their metabolism following activation supports the adoption of different effector functions, depending on the activating stimulus. Not only does this support increased energy demands and the upregulation of the correct pathways to provide an appropriate response to the activating stimulus, but metabolites themselves can act as signalling molecules. Metabolites of the Krebs cycle have been of particular interest, and roles for succinate and α-ketoglutarate (αKG) in macrophage differentiation have been described. This thesis aims to investigate whether specific Krebs cycle intermediates, or derivates thereof, can have immunomodulatory effects in macrophages.
This project has shown that a derivative of oxaloacetate, diethyl oxaloacetate (DOA), limits the production of pro-inflammatory mediators by lipopolysaccharide (LPS)-stimulated macrophages. DOA treated macrophages produce less of the precursor of interleukin-1β (pro-IL-1β), IL-10 and IL-6 in response to LPS. This is in part mediated by the activation of nuclear factor erythroid 2-related factor 2 (NRF2) by DOA. NRF2 is a key regulator of the antioxidant response, but it has also been shown to directly inhibit the expression of certain pro-inflammatory cytokines. DOA does not inhibit pro-IL-1β production in Nrf2-/- macrophages, and the inhibition of IL-10 by DOA is at least partially NRF2-dependent. DOA can also activate a negative regulator of secondary transcriptional responses to LPS, activating transcription factor 3 (ATF3), while inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor ζ (IκB-ζ), which is known to regulate secondary response genes. The effect of DOA on NRF2 is likely to be via its electrophilic properties.
An increase in the export of citrate from the mitochondria of classically activated macrophages has been shown to be important for the production of pro-inflammatory mediators. On the other hand, itaconate, produced from the conversion of mitochondrial citrate to itaconate by cis-aconitate decarboxylase 1 (ACOD1), is known to play a role in limiting inflammation in LPS-activated macrophages. The adenosine triphosphate (ATP) citrate lyase (ACLY) is responsible for the conversion of cytosolic citrate into oxaloacetate and acetyl-coenzyme A (acetyl-CoA). This study has shown that phosphorylation of ACLY is increased by LPS treatment in macrophages, indicating increased activity. Acetyl-CoA is a precursor for de novo fatty acid synthesis, but also an important cofactor for the acetylation of histone and non-histone proteins. Inhibition of ACLY by pharmacological means or genetic silencing resulted in a reduction of Il1b transcription, possibly indicating a role for histone acetylation in driving transcription of Il1b.
This study also showed that 2-hydroxyglutarate (2HG) was increased in LPS-activated macrophages, with abundance of both enantiomers elevated. L-2HG, but not D-2HG could promote the expression of IL-1β and the adoption of an inflammatory, highly glycolytic metabolic state. These changes are likely mediated thorough activation of hypoxia inducible factor-1α (HIF-1α), as L-2HG is known to inhibit the HIF prolyl hydroxylase (PHD) enzymes. Production of L-2HG is often linked to mitochondrial dysfunction, a known occurrence in M1 macrophages. Synthesis of L-2HG may be promoted by increased availability of the precursor metabolite, αKG. Expression of the enzyme responsible for L-2HG degradation, L-2HG dehydrogenase (L-2HGDH), was also decreased in LPS-stimulated macrophages and may therefore contribute to its accumulation.
In summary, this thesis shows that the oxaloacetate derivative, DOA, is an NRF2 activator, further indicates a role for citrate metabolism in inflammation via ACLY and identifies L-2HG as an activator of HIF-1α. This study therefore furthers our understanding of metabolic reprogramming of Krebs cycle in macrophages and indicates possible therapeutic targets which could have use in inflammatory diseases. | en |
