81 3 Responses of alveolar-like macrophages to lysine deacetylase inhibition plasticity of alveolar macrophages. In our case, we found more MMP12 in cells that have higher expression of anti-inflammatory genes. This could be one of the first steps leading to an attenuation of inflammatory responses through downregulation of recruitment of polymorphonuclear leukocytes in vivo (67). The only significantly differentially expressed proteins affected by both KDAC inhibitor treatments were Lims1 (LIM and senescent cell antigen-like-containing domain protein 1) and Ltv1 (low temperature viability protein 1), which were downregulated in both cases. Lims 1, also known as Pinch1, is a focal adhesion protein important in cellular processes like cell shape modulation, motility, and survival (68). Ltv1 is an assembly factor, that together with ribosomal proteins helps correct ribosome assembly (69). No direct correlation was found between Lims1 or Ltv1 expression and macrophage activity, indicating two possible proteins of interest to investigate further in alveolar macrophages. The aim of our studies was to investigate whether anti-inflammatory effects of KDAC inhibitors could be driven by metabolic changes in macrophages. Many studies have shown that when macrophages polarize to an anti-inflammatory or alternatively activated phenotype they rely on oxidative phosphorylation and on the beta-oxidation of fatty acids to produce ATP, while they rely on glycolysis when they polarize to a pro-inflammatory or classically activated phenotype. This dualistic view is now recognized as being overly simplistic (46,70), since it has recently been shown that tissue-resident alveolar macrophages do not rely on glycolysis to respond to pro-inflammatory stimuli (6). Using a multi-omics approach allowed us to confirm this recent finding in our model system and to study whether effects of KDAC inhibitors depend on metabolic changes in alveolar-like macrophages from many different angles. While we did not see any functional metabolic changes using extracellular flux analysis, treatment with LPS resulted in higher levels of three key intracellular metabolites of the TCA cycle. This finding is in line with proinflammatory metabolic reprogramming with a broken TCA cycle, represented by higher levels of succinate, and is also coupled with higher concentrations of malate and α-ketoglutarate. These two latter metabolites correlated to inflammation in macrophages by supporting the production of cytokines and other immune effector molecules in previous studies (49,50), suggesting that the changes in metabolism in alveolar macrophages may be too subtle to pick up with extracellular flux analysis. Mitochondrial respiration at the spare respiratory capacity level was slightly higher when cells were treated with KDAC inhibitor RGFP966. This deacetylase inhibitor clearly inhibited release of proinflammatory cytokines in LPS-treated macrophages and therefore the small but significant induction of mitochondrial respiration fits with this metabolic reprogramming (71,72). One limitation of our study was the
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