Background Genetic factors account for about fifty percent of the risk for alcoholism and alcohol dependence (AD) has been reported to be influenced by cannabinoid receptors (CBRs) and the endocannabinoid system (ECS). Previous studies have focused on cannabinoids and alcohol-related effects in the CNS; however, the role CBRs play on alcohol effects in the immune system has not been elucidated yet. Since alcohol can affect immune responses and have detrimental effects on immune cells such as dendritic cells (DCs), we hypothesize that alcohol can exert its effects on DCs by modulating changes in CBRs, which in turn can regulate important DC functions such as cytokine production. Methods Therefore, we studied the expression of CNR1 and CNR2, and the novel cannabinoid G protein-coupled receptor (GPCR) 55 (GPR55) in human monocyte-derived dendritic cells (MDDCs) from alcohol users. CNR1, CNR2, and GPR55 genes were measured by qRT-PCR and protein by flow cytometry. MDDCs from alcohol users show significantly higher levels of CNR2 and GPR55 compared to MDDCs from non-users. These findings were further confirmed using MDDCs treated with alcohol. Inflammatory cytokines were measured in EtOH-treated and non-treated cells by antibody array. Results Functional effects of CBRs on MDDCs were shown by CB2 and GPR55 siRNA transfection. Transfected EtOH-treated cells showed significantly higher levels of proinflammatory cytokine production as measured by IL-1β expression. Our results provide insights into alcohol mechanisms of DC regulation and show, for the first time, that alcohol is inducing CNR2 and GPR55 in human DCs.

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.

During human immunodeficiency virus (HIV) infection, alcohol has been known to induce inflammation while cannabinoids have been shown to have an anti-inflammatory role. For instance cannabinoids have been shown to reduce susceptibility to HIV-1 infection and attenuate HIV replication in macrophages. Recently, we demonstrated that alcohol induces cannabinoid receptors and regulates cytokine production by monocyte-derived dendritic cells (MDDC). However, the ability of alcohol and cannabinoids to alter MDDC function during HIV infection has not been clearly elucidated yet. In order to study the potential impact of alcohol and cannabinoids on differentiated MDDC infected with HIV, monocytes were cultured for 7 days with GM-CSF and IL-4, differentiated MDDC were infected with HIV-1Ba-L and treated with EtOH (0.1 and 0.2%), THC (5 and 10 μM), or JWH-015 (5 and 10 μM) for 4–7 days. HIV infection of MDDC was confirmed by p24 and Long Terminal Repeats (LTR) estimation. MDDC endocytosis assay and cytokine array profiles were measured to investigate the effects of HIV and substances of abuse on MDDC function. Our results show the HIV + EtOH treated MDDC had the highest levels of p24 production and expression when compared with the HIV positive controls and the cannabinoid treated cells. Although both cannabinoids, THC and JWH-015 had lower levels of p24 production and expression, the HIV + JWH-015 treated MDDC had the lowest levels of p24 when compared to the HIV + THC treated cells. In addition, MDDC endocytic function and cytokine production were also differentially altered after alcohol and cannabinoid treatments. Our results show a differential effect of alcohol and cannabinoids, which may provide insights into the divergent inflammatory role of alcohol and cannabinoids to modulate MDDC function in the context of HIV infection.

Previous reports on epigenetic mechanisms involved in alcohol abuse have focus on hepatic and neuronal regions, leaving the immune system and specifically monocyte-derived dendritic cells (MDDCs) understudied. Our lab has previously shown histone deacetylases are modulated in cells derived from alcohol users and after in vitro acute alcohol treatment of human MDDCs. In the current study, we developed a novel screening tool using matrix assisted laser desorption ionization-fourier transform-ion cyclotron resonance mass spectrometry (MALDI-FT-ICR MS) and single cell imaging flow cytometry to detect post-translational modifications (PTMs) in human MDDCs due to chronic alcohol exposure. Our results demonstrate, for the first time, in vitro chronic alcohol exposure of MDDCs modulates H3 and H4 and induces a significant increase in acetylation at H4K12 (H4K12ac). Moreover, the Tip60/HAT inhibitor, NU9056, was able to block EtOH-induced H4K12ac, enhancing the effect of EtOH on IL-15, RANTES, TGF-β1, and TNF-α cytokines while restoring MCP-2 levels, suggesting that H4K12ac may be playing a major role during inflammation and may serve as an inflammation regulator or a cellular stress response mechanism under chronic alcohol conditions.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

Due to the legalization of marijuana and the increased demand for cannabis and alcohol consumption, research efforts highlighting the biomedical consequences of the use of alcohol and cannabinoids are not only relevant to the substance abuse scientific field, but are also of public health interest. Moreover, an overview of the recent literature about alcohol and cannabinoids neuro-immunomodulatory effects highlighting their future therapeutic potentials will provide a significant contribution to science and medicine. Therefore, in the current review, we will first discuss briefly the prevalence of alcohol and marijuana abuse, followed by a discussion on the individual effects of alcohol and cannabinoids on the immune system; then, we will focus on the role of endocannabinoids on the alcohol-induced inflammatory effects. In addition, the review also incorporates cytokine array data obtained from human monocyte-derived dendritic cells, providing a different perspective on the alcohol and cannabinoid abuse divergent effects on cytokine production. The final section will highlight the therapeutic potential of cannabinoid receptors and the novel strategies to treat alcohol dependence as determined by in vitro, in vivo and clinical studies.

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Objective: The objective of this study was to understand whether histone deacetylase (HDACs) inhibitor Trichostatin A or TSA can block and/or reverse chronic alcohol exposure-induced ROS in human monocyte-derived dendritic cells (MDDCs). Additionally, since nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a known regulator of antioxidant responses, we studied the effects of alcohol and TSA on ROS production and modulation of Nrf2 by MDDCs. Methods: Intra-cellular, extra-cellular, and total ROS levels were measured in MDDCs treated chronically with alcohol (0.1 and 0.2 % EtOH) using 2’,7’-dichlorofluorescin diacetate (DCF-DA) followed by detection of ROS in microplate reader and imaging flow cytometer. Nrf2 expression was analyzed by qRT- PCR and western blot. In addition, NFE2L2 (Nrf2), class I HDAC genes HDAC1, HDAC2, and histone acetyltransferase genes KAT5 were analyzed in silico using the GeneMania prediction server. Results: Our results confirmed alcohol’s ability to increase intracellular ROS levels in MDDCs within minutes of treatment. Our findings have also demonstrated, for the first time, that TSA has a transient protective effect on MDDCs treated chronically with alcohol since the ability of TSA to reduce intracellular ROS levels is only detected up to 15 minutes post-chronic alcohol treatment with no significant protective effects by 10 hours. In addition, chronic alcohol treatment was able to increase the expression of the antioxidant regulator Nrf2 in a dose dependent manner, and the effect of the higher amount of alcohol (0.2%) on Nrf2 gene expression was significantly enhanced by TSA. Conclusion: This study demonstrates that TSA has a transient protective effect against ROS induced by chronic alcohol exposure of human MDDCs and chronic long-term exposure of MDDCs with alcohol and TSA induces cellular toxicity. It also highlights imaging flow cytometry as a novel tool to detect intracellular ROS levels. Overall, the effect of TSA might be mediated through Nrf2; however, further studies are needed to fully understand the molecular mechanisms.