There is abundant literature about different functional aspe

There is abundant literature about different functional aspects of the cannabinoid system (Badal et al., 2017, Balenga et al., 2011, Henderson-Redmond et al., 2016, Nair et al., 2015); however, not a great deal is known about the mechanism by which ethanol impacts its function in connection to the immune system. Previous reports have linked alcohol exposure to cannabinoid gene expression or function through epigenetic modifications (Parira et al., 2017, Subbanna et al., 2015). Previous in vitro studies with chronic and acute ethanol treatments showed that MDDCs exhibit an increase in global acetylation of H4K12 (Parira, Figueroa, et al., 2017). Additionally, GPR55 mRNA levels after acute ethanol treatment have been shown to be increased (Agudelo et al., 2013). Therefore, in the current study, we explored the possibility that H4K12 histone acetylation was linked to the increased expression of GPR55 in prostaglandin receptor treated acutely with ethanol. Remarkably, our findings demonstrate that ethanol-induced modulation of GPR55 expression by human MDDCs is accompanied by H4K12 acetylation. For instance, MDDCs treated acutely with ethanol at different concentrations showed a significant increase in expression of the GPR55 receptor when compared to control cells through confocal microscopy and imaging flow cytometry (Fig. 1), which is in agreement with previous findings using conventional flow cytometry in ethanol-treated cells (Agudelo, M. et al., 2013). MDDCs obtained from commercial buffy coats are challenging to use in studies because each biological sample comes with unique genetic and phenotypic characteristics, together with a fair amount of variation. Therefore, obtaining statistically significant differences between control and ethanol-treated cells in vitro gives additional validity to the study. Epigenetic modifications of intragenic chromatin regions have been shown to be essential for gene expression in mammalian cells. In some cases, they supersede the relevance of the upstream region of the transcription start site (Kobayashi et al., 2012, Shenker and Flanagan, 2012, Yan and Marsden, 2015, Yan et al., 2018). Therefore, we proceeded to determine through ChIP and real time qPCR that there is a significant enrichment of H4K12ac in the GPR55 gene region of the chromatin for cells acutely treated with ethanol, compared to non-treated cells (Fig. 2). Because histone acetylation events decrease the structural DNA-histone affinity, they create accessible regions in the chromatin that make it prone to transcription (Elvir, Duclot, Wang, & Kabbaj, 2017). With this in mind, we tested and established through pharmacological inhibition of Tip60 and siRNA transfection against Tip60 that the decrease in H4K12ac results in a significant decrease of GPR55 levels in MDDCs (Fig. 3), indicating that H4K12ac is involved in mediating the regulation of GPR55. However, the effects of NU9056 seem to be different from the Tip60 siRNA results, in the sense that NU9056 leads to a more significant decrease in GPR55 for non-ethanol treated cells. This might be because NU9056 can inhibit other histone acetyltransferases, although to a lesser extent than it does to Tip60 (Coffey et al., 2012), and the additional inhibition might have an effect on GPR55 expression as well. At the same time, the Tip60 siRNA silencing in ethanol-treated cells has a more significant effect than it does in non-ethanol treated cells, which suggests that Tip60 is essential for the GPR55 increase observed. These findings are relevant because GPR55 is abundantly expressed by immune cells (Zhou et al., 2016), and its epigenetic modulation through ethanol might have an impact on the functioning of the immune system. Furthermore, GPR55 has been shown to play a role in the regulation of inflammatory cytokines (Agudelo, M.et al., 2013; Stancic et al., 2015, Yang et al., 2016) produced by MDDCs, to be a multitasking regulator of cell signaling in different types of cells (Shore and Reggio, 2015, Stancic et al., 2015, Yang et al., 2016, Zhou et al., 2016), and to interact and modify the activity of other cannabinoid receptors (Balenga et al., 2011). Therefore, ethanol-induced GPR55 epigenetic regulation might be a defense mechanism to cope with cellular stress induced by ethanol.