150 Chapter 5 Although each of these techniques provides valuable information about macrophages, none can provide a comprehensive view on their own. Indeed, macrophage responses are highly dynamic and multifaceted. Using a combination of techniques would allow researchers to gain a more complete and nuanced understanding macrophage behavior under different conditions. By integrating data from many different assays, researchers can get a more accurate picture of macrophage activation, enabling the development of targeted therapies for inflammatory diseases and infections. KDACI’S ANTI-INFLAMMATORY EFFECT In Chapter 3 we have applied the above-mentioned approaches and knowledge to understand how lysine deacetylase inhibitors (KDACi) impact alveolar-like macrophages in the context of inflammation and metabolism. Our study confirmed the anti-inflammatory effects of MS275 (KDAC1,2, and 3 inhibitor) and RGFP966 (KDAC3 inhibitor) in combination with LPS exposure, previously described by Leus et al. [22,23]. In fact, we observed decreased TNF-α excretion in cells stimulated with LPS and treated with the inhibitors, and RGFP966 in particular, compared to cells that were only LPS-stimulated. Similar findings were found for IL-6. We hypothesized that the inhibition of lysine deacetylation, a post-translational modification, could affect, among other things, the stability and activity of metabolic enzymes, therefore inducing changes in macrophage metabolism. Unexpectedly, we observed minimal changes in macrophage metabolism despite reduced inflammation upon KDACi treatment. This suggests that alveolar-like macrophages might not rely heavily on metabolic reprogramming in response to inflammation, contrary to other macrophage types. Our proteomics analysis revealed that KDACi, especially KDAC3 inhibition, reduces inflammation by potentially enhancing protein ubiquitination. This modulation of ubiquitination could be a key driver of the anti-inflammatory effects of KDACi. Additionally, we noted that specific proteins, like INCA1 (Inhibitor Of CDK, Cyclin A1 Interacting Protein 1), JAM-A (junctional adhesion molecule A), and MMP12 (macrophage metalloelastase 12), were differentially expressed in response to KDACi treatment, suggesting their roles in macrophage behavior. Our study provides insights into the complex interplay between KDACi, inflammation, and metabolism in alveolar-like macrophages. In summary, KDACi, particularly KDAC3 inhibition, dampens inflammation in alveolar-like macrophages, possibly through enhancing ubiquitination, without drastically altering their metabolism. This highlights the potential of targeting
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