63 3 Responses of alveolar-like macrophages to lysine deacetylase inhibition lung inflammation in smoke-exposed mice and inflammatory mediator production in lipopolysaccharide (LPS)-exposed lung slices (20,21). KDACs are executors of epigenetic mechanisms by removing acetyl groups from the ϵ-amino group of lysine residues in target proteins, thereby altering the way these proteins interact with other proteins/nucleic acids (22) or altering their activity (23). Acetyl-CoA, an important metabolic intermediate, is the substrate of lysine acetyltransferases (KATs) that acetylate proteins. Therefore, altered levels of acetylCoA during metabolic reprogramming of macrophages may alter the level of lysine acetylation (23), which may subsequently affect gene expression. The focus of this study was to investigate whether lysine deacetylase inhibitors inhibit activation of primary macrophages by changing metabolic processes in these cells. To this end, we used self-propagating murine alveolar-like macrophages derived from fetal liver monocytes, a well-established model for alveolar macrophages (24–26). To investigate the relationship between anti-inflammatory effects of KDAC inhibition and metabolic processes in alveolar-like macrophages, we studied metabolites, metabolic enzymes, and amino acids by targeted liquid chromatography–mass spectrometry (LC-MS), gas chromatography–mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC) with fluorescence detection, as well as cellular responses by extracellular flux analysis. MATERIALS AND METHODS Cell culture Self-propagating murine alveolar-like macrophages (a kind gift from dr. G.Fejer, Plymouth University, UK (27)) isolated from fetal livers were cultured in plastic tissue culture flasks (Costar Europe) at 37⁰C with 5% CO2/95% air in RPMI 1640 medium containing GlutaMAXTM (Gibco® ; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco®), and 20 ng/mL murine granulocyte-macrophage colony-stimulating factor (PeproTech; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Once a week, cells were detached with 1mM EDTA (Merck, Darmstadt, Germany) and reseeded at a density of 1,5 x 106 cells/ mL. Cell culture medium was replaced after 4 days. For experiments, macrophages were used between passage 3 and 15.
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