144 Chapter 5 of these metabolites, as reported previously [4]. In our system, succinate and malate concentrations increased and this has been reported to happen in pro-inflammatory macrophages due to the inactivation of the enzyme succinate dehydrogenase (SDH). SDH is a component of the electron transport chain that normally converts succinate to fumarate. This reduction in SDH activity leads to the accumulation of succinate and malate, which are used in several ways to support immune responses [16]. Succinate is a key signaling molecule that activates the transcription factor HIF1α, which in turn promotes the expression of pro-inflammatory genes encoding for interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) [17]. Increased levels of α-ketoglutarate in pro-inflammatory macrophages due to glutamine anaplerosis, when glutamine is converted via α-ketoglutarate into succinate through glutaminolysis or through an upregulated γ-aminobutyric acid (GABA) shunt. α-Ketoglutarate is a key metabolite that feeds into several biosynthetic pathways, including amino acids and nucleotides synthesis, as well as ATP production. In pro-inflammatory macrophages, the higher α-ketoglutarate levels may support the production of cytokines and other immune effector molecules [18]. However, for the other TCA breakpoint, we did not find any evidence in our system, as we did not observe an accumulation of citrate and/ or an increased glycolytic function, associated with the ``classically activated`` metabolic reprogramming [19]. This is in line with recently published data showing that tissue-resident alveolar macrophages do not rely on glycolysis to respond to LPS as a pro-inflammatory stimulus [20,21]. Since stimulation of alveolar-like macrophages with LPS resulted in higher production of the pro-inflammatory cytokine TNF-α, which was inhibited by cotreatment with two different lysine deacetylase inhibitors, we wanted to investigate the role of metabolic changes in the anti-inflammatory response of these cells. Treatment with the inhibitors also resulted in higher levels of the antiinflammatory cytokine IL-10. This finding is in agreement with earlier results showing that these inhibitors have an anti-inflammatory effect in smoke-exposed mice and in LPS-exposed lung slices [22,23] (further discussed in paragraph “KDACi’s anti-inflammatory effect”). The lysine deacetylase inhibitor (KDACi) RGFP966 had a significant effect on mitochondrial respiration by increasing the spare respiratory capacity as well as the level of maximal respiration compared to treatment with LPS alone. This deacetylase inhibitor, which had a stronger anti-inflammatory effect at the cytokine level among the KDACis used on LPS-treated macrophages, induced macrophage repolarization towards an anti-inflammatory phenotype. This phenomenon is thought to be accompanied by metabolic reprogramming, when macrophages rely more on oxidative phosphorylation, as demonstrated by the higher mitochondrial
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