br Introduction Lysine acetylation is an

Introduction Lysine acetylation is an important post-translational modification that is found on numerous cellular proteins, including both histone and non-histone proteins [1], [2]. Dynamic acetylation and deacetylation is regulated by enzymes called histone acetyltransferases (HATs) as writers and histone deacetylases (HDACs) as erasers. HAT and HDAC activity, and the balance between these activities, plays a crucial role in the regulation of multiple cellular processes [3], [4]. Moreover, HAT and HDAC activity, and dysregulation of the balance between the activities of these enzymes, has been implicated in multiple pathological conditions such as inflammatory disease and cancer, indicating that these enzymes represent potential drug targets in these diseases [5], [6], [7], [8]. The importance of the activity of these enzymes in diseases is exemplified by the inflammatory lung disease d mannitol in which the balance between HAT and HDAC activity is dysregulated through a shift towards increased HAT activity [9]. Chronic obstructive pulmonary disease (COPD), another inflammatory lung disease, is characterized by a loss of HDAC2 expression and activity, which also implies a shift in the balance between HAT and HDAC activity [9]. This suggests that restoring the balance between HAT and HDAC activity could be an alternative therapeutic strategy for inflammatory lung diseases such as asthma and COPD. There is an unmet need for this since severe asthmatics display glucocorticoid resistance [10], and since next to this, the effectivity of glucocorticoids in COPD patients is subject to debate [11], [12]. We hypothesize that restoring the balance between HAT and HDAC activity using small molecule inhibitors of HATs may be a starting point for novel treatments for inflammatory lung diseases such as asthma and COPD. Zooming in on the HAT enzymes, these are a disparate group of enzymes from which most isoenzymes can be assigned to 5 main families based on their primary structure homology. Three families that have been studied extensively are the GNAT (GCN5-related N-acetyltransferase) family, represented by KAT2A (also known as GCN5; general control nonderepressible 5) and KAT2B (also known as PCAF; p300/CBP associated factor); the p300/CBP family, including KAT3A (also known as CBP; CREB-binding protein) and KAT3B (also known as p300); and the MYST family including KAT5 (also known as Tip60) and KAT8 (also known as MOF) [4]. Over recent years several small molecule HAT inhibitors (HATi) have been described [6], targeting different HAT enzymes. This gives rise to the possibility of studying if these small molecule HATi have potential to achieve inhibition of expression of specific pro-inflammatory genes in model systems for inflammatory lung diseases such as asthma and COPD. The HATi Anacardic Acid (AA), which is a 6-alkylsalicylate, has been reported to inhibit the HATs KAT8, KAT5, KAT2B and KAT3B [13], [14]. Interestingly, AA decreased the expression of IL-4, IL-5 and IL-13 in T cells isolated from mice challenged with ovalbumin to model allergic asthma. Upon re-administering these T cells to mice, the balance between HDAC and HAT activities were changed in lung tissue towards more HDAC activity [15]. Furthermore, in a mouse model, AA was found to ameliorate lung damage which was induced by exposure of the mice to diesel exhaust particles. This effect was attributed to reduced levels of neutrophils in the lung parenchyma and reduced TNF-α levels in the BALF supernatant [16]. These studies demonstrate that 6-alkylsalicylates which inhibit HATs have potential for the treatment of inflammatory lung diseases. Using AA as a starting point our group developed the 6-alkylsalicylate MG149 [14] (Fig. 1) in which the 15 carbon atom 6-alkyl tail is replaced by a 4-heptylphenethyl substituent, providing a molecule with reduced lipophilicity and flexibility. Next to this, in terms of selectivity, MG149 shows less inhibition for the HATs PCAF and p300 compared to AA measured at concentrations of 200 μM, while retaining inhibitory potency for the MYST type HATs. We envision that this molecule provides a good starting point to investigate the potential of HAT inhibition for inhibition of inflammatory responses in model systems for inflammatory lung diseases. In this study we set out to investigate the potential anti-inflammatory effects of the 6-alkylsalicylate MG149 in model systems for inflammatory lung diseases such as asthma and COPD in connection to its HAT inhibitory activity. As a model system for inflammatory lung diseases murine precision-cut lung slices (PCLS) were selected. The lung tissue structure and cell-cell interactions are maintained in PCLS [17], thus providing a relevant ex-vivo model for lung inflammation. An advantage associated with the use of these type of organ slices is that the amount of required experimental animals can be reduced [18]. Since promoting roles for lipopolysaccharide (LPS) and interferon gamma (IFNγ) have been described in asthma and COPD, as reviewed by Boorsma et al. [19], a combined stimulus of LPS and IFNγ was selected as an inflammatory stimulus in PCLS.