79 3 Responses of alveolar-like macrophages to lysine deacetylase inhibition DISCUSSION Our main aim was to investigate if anti-inflammatory effects of lysine deacetylase inhibitors in alveolar-like macrophages were associated with metabolic changes in these cells to elucidate their mechanism of action. We focused on macrophage metabolism as metabolic adaptations are functionally important in many types of macrophages and lysine acetylation is closely associated with cell metabolism. To our knowledge, this is the first study to investigate the effects of lysine deacetylase inhibitors in this context using a multi-omics approach. Our finding that alveolarlike macrophage metabolism is mostly unchanged after a pro-inflammatory stimulus like LPS was rather unexpected. It suggests that, even though metabolic reprogramming is crucial in many other types of macrophages to respond to pro- and anti-inflammatory stimuli, alveolar-like macrophages do not seem to rely on this mechanism. This is in agreement with data reported by Woods et al. (6). Based on our proteomics results, we hypothesize that other post-translational modifications, such as ubiquitination, may be potential drivers of the anti-inflammatory effects of KDAC inhibitors. Our data illustrate that a multi-omics approach is necessary to discover how a complex cell model interacts with its environment. More detailed studies investigating ubiquitination as a potential driver of KDAC inhibition may help developing novel anti-inflammatory drugs for difficult to treat diseases such as COPD. Leus et al (20,21) previously showed that KDAC inhibitors MS275 and RGFP966 attenuated smoke- and LPS-induced inflammation in lung tissue. Our results confirm that these inhibitors exert similar anti-inflammatory effects in alveolar-like macrophages and they suggest further that KDAC3 inhibition by RGFP966 results in a more pronounced inhibitory effect than inhibiting KDAC1, 2, and 3 by MS275 in LPS-stimulated macrophages. This latter inhibitor only induced significantly higher expression of SOCS3 mRNA, suggesting a time lag compared to RGFP966, that already resulted in differential expression at the cytokine secretion level. This may be related to the lower specificity of MS275, a fact that is corroborated by our finding that MS275 treatment induced more changes to the proteome than RGFP966. In fact, in a cell-free system MS275 inhibited KDAC1, 2, and 3 (IC50 0.228, 0.364, and 0.744 μM, respectively), while RGFP966 only selectively inhibited KDAC3 (IC50 2.686 μM) (51), and similar values were obtained in RAW264.7 macrophages (52). Our proteomics results in conjunction with subsequent pathway analyses suggest that MS275 and RGFP966 mediate their anti-inflammatory effects by increasing protein ubiquitination and that this may be a potential driver of the anti-inflammatory effects of KDAC inhibitors. Protein ubiquitination is a posttranslational modification process that plays a crucial role in maintaining cellular
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