br Conclusions br Competing interests br Introduction Ethano

Conclusions
Competing interests
Introduction Ethanol exposure has been linked to changes in the pattern of histone acetylation and deacetylation through histone acetyltransferases (HATs) and histone deacetylases (HDACs) in the 3-Deazaneplanocin and in peripheral tissues, leading to ethanol dependence-related consequences in different organisms (Krishnan, Sakharkar, Teppen, Berkel, & Pandey, 2014). Some aspects regarding the connection between ethanol effects and the endocannabinoid system have been studied and reviewed previously (Basavarajappa, 2015, Henderson-Redmond et al., 2016, Nair et al., 2015, Parira et al., 2017). However, a significant knowledge gap still remains when it comes to understanding the role of endocannabinoid signaling in mediating ethanol effects in innate immune cells such as dendritic cells. The cannabinoid receptors CB1 and CB2 have been previously studied in the aspect of ethanol abuse and have been reviewed in terms of immunomodulatory effects (Nair et al., 2015). Most recently, studies on the novel cannabinoid receptor GPR55 have recently started to gain momentum with its emergence as a potential therapeutic drug target (Shore & Reggio, 2015). Our group has previously shown that cannabinoid receptors CB2 and GPR55 are upregulated in dendritic cells isolated from ethanol users and in in vitro cultures of human monocyte-derived dendritic cells (MDDCs) treated acutely with ethanol (Agudelo et al., 2013; Parira, Figueroa, et al., 2017). We have also recently found that chronic ethanol treatments in human MDDCs increased acetylation at histone (H) 4 lysine (K) 12 (Parira, Figueroa, et al., 2017). With those findings in mind, the aim of the current study is to determine whether there is any relevant association between the observed histone hyperacetylation and the regulation of the novel cannabinoid receptor GPR55, with a bigger aim of elucidating a key mechanism of action of ethanol on human MDDCs. To study the mechanism associated with ethanol upregulation of GPR55, human MDDCs were treated with ethanol for 24 h. Upregulation of protein expression levels for GPR55 was then confirmed in ethanol-treated MDDCs through confocal microscopy. Later, chromatin immunoprecipitation (ChIP) coupled with quantitative PCR gene expression analysis was carried out for the detection of GPR55, and the results confirmed that H4K12ac was enriched at the GPR55 gene in ethanol-treated MDDCs but not in control cells. Following ChIP-qPCR studies and in order to understand the nature of association between H4K12ac and GPR55, we also carried out pharmacological inhibition or siRNA silencing of histone acetyltransferase Tip60, which has been shown to be responsible for H4K12 acetylation (Grézy, Chevillard-Briet, Trouche, & Escaffit, 2016). Surprisingly, the results demonstrated, for the first time, that in the presence of ethanol, the upregulation of GPR55 expression is accompanied by H4K12 acetylation, which might have a significant effect on the ability of this innate immune system's cells to cope with cellular stress induced by ethanol. However, the causality of ethanol regulation of H4K12ac in GPR55 expression changes still lacks further elucidation; therefore, additional experimental approaches to confirm a significant causality between H4K12 acetylation and ethanol regulation of GPR55 are currently in progress in our lab. GPR55 is a key component of the endocannabinoid system (ECS), affecting numerous aspects of immunity such as cytokine levels, immune cell modulation, and transcription factor activation (Zhou, Burkovskiy, Yang, Sardinha, & Lehmann, 2016). Therefore, studies elucidating the regulation of GPR55 may contribute significantly to the pool of knowledge regarding ethanol abuse by providing a new perspective on the ethanol-mediated epigenetic mechanisms involved in the inflammatory consequences of ethanol abuse.
Materials and methods
Results
Discussion Exogenous substances such as drugs of addiction and ethanol shape the social behavior of millions of people and affect how well our immune cells can face challenges (Ratna & Mandrekar, 2017). One category of those substances is cannabinoids, and they have been shown to be an important element for immune regulation (Abidi et al., 2018, Simon et al., 2016, Tanasescu and Constantinescu, 2010). Most recent preliminary findings investigating the relationships between alcohol consumption, cannabis use, and circulating cytokines have demonstrated that cannabinoid compounds may serve to mitigate inflammation associated with alcohol use (Karoly, Bidwell, Mueller, & Hutchison, 2018). In addition, the cannabinoid system, through its network of receptors, has been shown to be modulated by ethanol (Agudelo et al., 2013, Khatri et al., 2018, Parira et al., 2017, Varodayan et al., 2017). Therefore, the present work was aimed at uncovering the molecular mechanism of ethanol-mediated immune modulation of MDDCs through GPR55.