Epigenetic Mechanisms Regulating the Functional Effects of Chronic Alcohol Exposure of Human Monocyte-derived Dendritic Cells

The effects of alcohol abuse are multi-dimensional since alcohol is widely known to affect both the innate and adaptive immune systems. Recently, epigenetics has come into focus and has been implicated in many diseases as well as substance abuse disorders. Therefore, research efforts of understanding the epigenetic mechanisms underlying substance abuse effects including alcohol abuse have become more predominant. In our laboratory, we have studied different epigenetic changes induced by alcohol exposure including regulation of histone deacetylases (HDACs), histone quantity, and histone modifications such as acetylation and deacetylation. We have observed differential effects of acute and chronic alcohol exposure in human monocyte-derived dendritic cells (MDDCs) wherein our laboratory previously found that HDACs were modulated in MDDCs treated acutely with alcohol in vitro, and in MDDCs from alcohol users. Our previous work has also demonstrated that alcohol consumption affects the dendritic cell function by modulating inflammatory markers and cannabinoid receptors such as CB2 and GPR55 through epigenetic modifications. For instance, chronic alcohol exposure upregulates histone (H) 4 acetylation at lysine 12 (H4k12ac) and acute alcohol effects on histone acetylation are associated with an increase in GPR55 expression. The hypothesis of the study is that chronic alcohol modulates human MDDC function through epigenetic mechanisms. Therefore, the primary objective of this research project is to elucidate novel pathways involving histone post-translational modifications due to chronic alcohol exposure in human dendritic cells. For this study, monocytes isolated from commercially available human buffy coats were differentiated into MDDCs, which were treated with chronic alcohol levels of 0.1 % (100mg/dL) and 0.2 % (200mg/dL) for 5 days in the presence or absence of the histone acetyltransferase inhibitor NU9056 (50nM) or the GPR55 antagonist CID16020046 (5µM). Results showed that chronic alcohol levels upregulated H4K12ac in MDDCs and this was associated with a concomitant increase in GPR55 gene and protein expression. Further, NU9056 and CID16020046 were able to reduce alcohol-induced inflammatory chemokine MCP-2 and reactive oxygen species production indicating that H4K12ac may be an inflammation and oxidative stress regulator. Additionally, NU9056 and CID16020046 could potentially reduce alcohol-induced inflammation and serve as potential therapeutic targets for alcohol use disorders

Profile of Class I Histone Deacetylases (HDAC) by Human Dendritic Cells after Alcohol Consumption and In Vitro Alcohol Treatment and Their Implication in Oxidative Stress: Role of HDAC Inhibitors Trichostatin A and Mocetinostat

Epigenetic mechanisms have been shown to play a role in alcohol use disorders (AUDs) and may prove to be valuable therapeutic targets. However, the involvement of histone deacetylases (HDACs) on alcohol-induced oxidative stress of human primary monocyte-derived dendritic cells (MDDCs) has not been elucidated. In the current study, we took a novel approach combining ex vivo, in vitro and in silico analyses to elucidate the mechanisms of alcohol-induced oxidative stress and role of HDACs in the periphery. ex vivo and in vitro analyses of alcohol-modulation of class I HDACs and activity by MDDCs from self-reported alcohol users and non-alcohol users was performed. Additionally, MDDCs treated with alcohol were assessed using qRT-PCR, western blot, and fluorometric assay. The functional effects of alcohol-induce oxidative stress were measured in vitro using PCR array and in silico using gene expression network analysis. Our findings show, for the first time, that MDDCs from self-reported alcohol users have higher levels of class I HDACs compare to controls and alcohol treatment in vitro differentially modulates HDACs expression. Further, HDAC inhibitors (HDACi) blocked alcohol-induction of class I HDACs and modulated alcohol-induced oxidative stress related genes expressed by MDDCs. In silico analysis revealed new target genes and pathways on the mode of action of alcohol and HDACi. Findings elucidating the ability of alcohol to modulate class I HDACs may be useful for the treatment of alcohol-induced oxidative damage and may delineate new potential immune-modulatory mechanisms

<i>in silico</i> Oxidative Stress and Antioxidant Defense Gene Network Interactions with Class I HDACs.

<p>Non-specific interactions between <b>A)</b> antioxidant, <b>B)</b> ROS metabolism genes, <b>C)</b> all class I HDACs. Tissue-specific interactions between <b>D)</b> HDAC1, HDAC2 and HDAC3 with PCR array gene targets (CSDE1, CYBA, SGK2, TXNDC2) and individual HDACs interactions including <b>E)</b> HDAC1, <b>F)</b> HDAC2, and <b>G)</b> HDAC3. Interactions are color-coded by down-regulation, up-regulation, regulation, co-expression, chemical modification, physical interaction, predicted protein interaction, and predicted TFactor regulation.</p

Effects of Alcohol and/or HDACi on Oxidative Stress and Antioxidant Defense Genes.

<p>Effects of Alcohol and/or HDACi on Oxidative Stress and Antioxidant Defense Genes.</p

Alcohol Induces Protein Levels of HDACs <i>ex vivo</i> in MDDCs from Alcohol Users.

<p><b>A)</b> Representative blot is shown for MDDC from alcohol users and non-users. Whole cell lysates (20 μg) were used for western blot analysis. <b>B)</b> Optical densities (OD) were analyzed using Image J software. Protein quantification is expressed as percentage of control ± SEM of ten OD readings per HDAC. p≤0.05 is considered significant.</p

Effects of Alcohol or HDACi (TSA and MGCD0103) on MDDCs Viability.

<p>MDDCs were treated for 24 and 48 hours with <b>A and B</b>) different percentages of alcohol (EtOH 0.05–0.4%) and <b>C and D</b>) different concentrations of HDACi: TSA (50-100nM) and MGCD0103 (1–6μM). Viability was assessed by dye exclusion method using trypan blue. Data are expressed as averages of percent viable cells normalized to control ± SE, (n = 7).</p

<i>in vitro</i> HDACi Treatment Significantly Inhibits the Alcohol Effect in all Class I HDACs.

<p><b>A and F)</b> Representative blot is shown for MDDCs pre-treated with TSA (50 nM) or MGCD0103 (3 μM) and/or treated with alcohol (0.2% EtOH, 0.2 g/dL). Total protein lysates (20 μg) were used for western blot analysis. <b>B-E and G-J)</b> Protein quantification of two independent experiments. Optical density levels were analyzed using Image J software. Data are expressed as percentage of control ± SEM of two OD readings per HDAC; p≤0.05 is considered significant.</p

Representative Scatter Plot Analysis of the Changes in Oxidative Stress and Antioxidant Defense Gene Expression by MDDCs Treated with EtOH and/or HDACi.

<p>Pair wise comparison of untreated and treated MDDCs with <b>A)</b> 0.2% EtOH (0.2g/dL) <b>B)</b> TSA (50 nM), <b>C)</b> TSA + EtOH, <b>D)</b> MGCD0103 (3 μM), and <b>E)</b> MGCD0103 + EtOH was performed by scatter plot analysis after running arrays by qRT-PCR. Data were analyzed using GeneGlobe Data Analysis.</p