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Background: Alcohol use disorder is a significant societal and medical burden that is associated with both organ pathology and addiction. Excessive alcohol use results in neuroinflammation characterized by activation of the inflammasome, a multiprotein complex, and IL‐1β increase in the brain. Recent studies suggest that inflammation could contribute to alcohol addiction. Here, we targeted components of the NOD‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome cascade, which senses and responds to immunologic stimuli, to determine whether NLRP3 inhibition modulates alcohol consumption. Methods: C57BL/6J male and female mice were provided a 2‐bottle choice of alcohol at increasing concentrations (3, 6, 9, and 12%, 4 days each) or water, and some were treated with daily injections of an NLRP3 inhibitor (MCC950), a caspase‐1 inhibitor (VX765), IL‐1 receptor antagonist (IL‐1ra; anakinra), or vehicle injection. Results: Treatment with VX765, MCC950, and IL‐1ra significantly reduced alcohol consumption and preference in female mice (p < 0.05). Treatment with MCC950 and IL‐1ra reduced alcohol consumption, while IL‐1ra reduced alcohol preference in male mice (p < 0.05). VX765 did not affect alcohol consumption or preference in male mice. Conclusions: These findings highlight gender differences in alcohol preference and demonstrate that inhibition of different steps in inflammasome signaling can reduce alcohol consumption in females. Inhibition of NLRP3 inflammasome activation and the inflammasome‐IL‐1β cascade opens novel insights into the development of new therapies to address alcohol use disorder in an era of targeted and precision medicine. [ABSTRACT FROM AUTHOR]

Background: Binge drinking is increasingly recognized as an important cause of liver disease with limited therapeutic options for patients. Binge alcohol use, similar to chronic alcohol consumption, induces numerous deregulated signaling events that drive liver damage, steatosis, and inflammation. In this article, we evaluated the role of spleen tyrosine kinase (SYK), which modulates numerous signaling events previously identified linked in the development alcohol-induced liver pathology. Methods: A 3-day alcohol binge was administered to C57BL/6 female mice, and features of alcoholic liver disease were assessed. Some mice were treated daily with intraperitoneal injections of a SYK inhibitor (R406; 5 to 10 mg/kg body weight) or drug vehicle control. Liver and serum samples were collected and were assessed by Western blotting, biochemical, ELISA, electrophoretic mobility shift assays, real-time quantitative polymerase chain reaction, and histopathological analysis. Results: We found that binge drinking induced significant SYK activation (SYKY525/526) with no change in total SYK expression in the liver. Functional inhibition of SYK activation using a potent SYK inhibitor, R406, was associated with a significant decrease in alcohol-induced hepatic inflammation as demonstrated by decreased phospho-nuclear factor kappa beta (NF-κB) p65, NF-κB nuclear binding, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 mRNA in the liver. Compared to vehicle controls, SYK inhibitor treatment decreased alcohol binge-induced hepatocyte injury indicated by histology and serum alanine aminotransferase. Strikingly, SYK inhibitor treatment also resulted in a significant reduction in alcohol-induced liver steatosis. Conclusions: Our novel observations demonstrate the role of SYK, activation in the pathomechanism of binge drinking-induced liver disease highlighting SYK a potential multifaceted therapeutic target. [ABSTRACT FROM AUTHOR]

Background Clinical studies demonstrate synergistic liver damage by alcohol and hepatitis C virus ( HCV); however, the mechanisms by which alcohol promotes HCV infection remain obscure. The liver-specific micro RNA-122 (miR-122) regulates HCV replication and expression of host genes, including Cyclin G1. Here, we hypothesized that alcohol regulates mi R-122 expression and thereby modulates HCV RNA replication. Methods The J6/ JFH/ Huh-7.5 model of HCV infection was used in this study. Real-time quantitative polymerase chain reaction, Western blotting, electrophoretic mobility shift assay, and confocal microscopy were used for experimental analysis. Results We found that acute alcohol exposure (25 mM) significantly increased intracellular HCV RNA as well as mi R-122 levels in Huh-7.5 and Huh-7.5/ CYP2 E1 expressing cells in the presence and absence of J6/ JFH- HCV infection. Expression of the mi R-122 target, Cyclin G1, was inhibited by alcohol both in J6/ JFH-infected and uninfected Huh-7.5 cells. The use of a mi R-122 inhibitor increased Cyclin G1 expression and prevented the alcohol-induced increase in HCV RNA and protein levels, suggesting a mechanistic role for alcohol-induced mi R122 in HCV replication. We discovered that si RNA-mediated silencing of Cyclin G1 significantly increased intracellular HCV RNA levels compared with controls, suggesting a mechanistic role for Cyclin G1 in HCV replication. Alcohol-induced increase in mi R-122 was associated with increased nuclear translocation and DNA binding of the nuclear regulatory factor-κ B and could be prevented by NF-κ B inhibition. Conclusions Our novel data indicate a mi R-122-mediated mechanism for alcohol increasing HCV RNA replication. We show for the first time that Cyclin G1, a mi R-122 target gene, has regulatory effects on HCV replication and that alcohol increases HCV replication by regulating mi R-122 and Cyclin G1. [ABSTRACT FROM AUTHOR]

Background Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-κB in monocytes in the presence of ongoing IκBα degradation, suggesting regulation of NF-κB activation downstream of IκBα degradation. DNA binding of NF-κB has been suggested to be regulated by other nuclear regulatory factors, including the glucocorticoid receptor (GR). Here, we show for the first time that acute alcohol (25 mM) exposure modulates GR activation in monocytes. Methods Human peripheral blood monocytes were treated with lipopolysaccharide (LPS) in the presence or absence of alcohol (25 mM) for 1 hour. Nuclear GR levels were estimated by Western blotting and NFκB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 μM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IκBα mRNA known to be induced by GR/GRE activation was studied in total RNA extracts by the SuperArray method (SuperArray Inc., Bethesda, MD). Results LPS is a potent inducer of GR nuclear translocation and GR binding to the glucocorticoid response element (GRE). Acute alcohol treatment both induced ( p < 0.05) and augmented ( p < 0.05) LPS-stimulated GR nuclear levels. However, alcohol inhibits LPS-induced (nonligand bound) GR/GRE binding activity in monocytes. This inhibition of GR transactivation by alcohol was further confirmed by decreased expression (40%) of a target gene, IκBα. Thus, alcohol treatment increases nonligand-bound nuclear GR, but inhibits its transactivation function. Ligand-induced GR/GRE binding was decreased in alcohol-treated monocytes. Inhibition of ligand-induced GR/GRE binding by alcohol exposure is likely due to cytoplasmic retention of the GR. Conclusions Our results show that acute alcohol exposure inhibits GR in monocytes by differently affecting ligand- and nonligand-induced GR nuclear translocation. These data also suggest that acute alcohol regulates GR activation in monocytes concomitant to inhibition of NF-κB activation. [ABSTRACT FROM AUTHOR]

Background Nuclear translocation and DNA binding of the nuclear factor κB (NF-κB) is an early event in inflammatory cell activation in response to stimulation with bacterial components or cytokines. Cell activation via different receptors culminates in a common pathway leading to NF-κB activation and proinflammatory cytokine induction. We have previously shown that acute alcohol inhibits NF-κB activation by lipopolysaccharide (LPS) in human monocytes. Here we investigated whether acute alcohol treatment of human monocytes also inhibits NF-κB when induced through activation of the interleukin (IL)-1 or tumor necrosis factor (TNF) receptors. Methods Human peripheral blood monocytes were treated with LPS, TNFα, and IL-1β in the presence or absence of 25mM alcohol for 1 hr. NF-κB activation was determined by electrophoretic mobility shift assays using nuclear extracts. Inhibitory κBα (IκBα) was estimated by Western blotting in cytoplasmic extracts. Chinese hamster ovary cells expressing human CD14 were treated with LPS in the presence or absence of alcohol to study NF-κB and IκBα regulation. Results Our results indicate that acute alcohol inhibits IL-1β- and TNFα-induced NF-κB activation. We further show in CD14/toll-like receptor 4-expressing Chinese hamster ovary cells the specificity of alcohol-mediated inhibition of NF-κB via the toll-like receptor 4/CD14 receptors. Inhibition of NF-κB by acute alcohol was concomitant with decreased levels of the IκBα molecule in the cytoplasm of LPS, IL-1, and TNFα-activated monocytes. Conclusions These data suggest a unique, IκBα-independent pathway for the inhibition of NF-κB activation by acute alcohol in monocytes. Universal inhibition of NF-κB by acute alcohol via these various receptor systems suggests a target for the effects of alcohol in the NF-κB activation cascade that is downstream from IκBα degradation. Further, these results demonstrate that acute alcohol is a potent inhibitor of NF-κB activation by mediators of early (LPS) or late (IL-1, TNFα) stages of inflammation in monocytes. [ABSTRACT FROM AUTHOR]

Background: Immunosuppression associated with chronic alcohol use is characterized by reduced antigen-specific T-cell response and impaired delayed type hypersensitivity. Increasing evidence suggests in chronic alcohol consumption models that reduced antigen-specific T-cell proliferation is due to insufficient accessory cell function. Accessory cell function, a critical step in recognition of viral antigens, is reduced in chronic hepatitis C. The severity of hepatitis C is increased by alcohol consumption. Thus, we investigated the effects of alcohol consumption on accessory cell activity of monocytes in supporting alloreactive T-cell proliferation. Methods: Alloreactive T-cell proliferation was evaluated in a one-way mixed lymphocyte reaction (MLR). Mononuclear cells were isolated by Ficoll density gradient and monocytes by adherence. Alcohol (0.8 g/kg body weight, an equivalent of approximately three drinks) was given to nonalcohol-consuming individuals and blood samples were collected before, 4 hr, or 18 hr after alcohol consumption. Alcohol in vitro was administered at concentrations of 25-100 mM. Results: T-cell proliferation in MLR was significantly reduced in the presence of physiologically relevant concentrations of alcohol in vitro (25-100 mM ethanol) ( p < 0.05). In vivo alcohol consumption also depressed proliferation in the MLR when stimulator cells were obtained 4 hr after alcohol consumption. MLR was not decreased, however, in the presence of alcohol-exposed responder cells and normal stimulator cells, suggesting that the accessory cell population and not T cells are affected by alcohol. Decreased accessory cell function was further evidenced by reduced superantigen-induced (SEB) but not mitogen-induced (PHA) T-cell proliferation in samples obtained 18 hr after alcohol intake (35% reduction). Reduced accessory cell function was not due to changes in surface expression of monocyte costimulatory molecules (HLA class I, HLA-DR, CD80, CD86, CD40). We found reduced IFNγ, elevated IL-10, and unchanged IL-4 levels during T-cell proliferation in samples obtained 18 hr after alcohol consumption. Acute alcohol treatment resulted in increased IL-13 in the MLR. Conclusion: These data suggest that even on one occasion moderate alcohol intake can reduce allostimulatory T-cell activation via decreasing accessory cell function. Increased IL-10 and IL-13 plus the reduced IFNγ production after acute alcohol use are likely to contribute to both the reduced T-cell proliferation and monocyte accessory cell function. These accessory cell mediated defects in T-cell activation may result in impaired antiviral and antitumor immunity after moderate acute alcohol use. [ABSTRACT FROM AUTHOR]

Background: The mechanisms for decreased therapeutic response to IFNα in chronic hepatitis C patients with alcohol are unknown. We investigated the hypothesis that IFNα and alcohol regulate cells both in the liver parenchyma and the immune system. Methods: We used the hepatocellular carcinoma cells (HepG2) to determine if IFNα (500-10,000 U/ml) or ethanol (25-100 mM) modulates NF-kB activation alone or in combination with TNFα (0.1-20 μg/ml) as determined in electromobility gel shift assays. IkB levels were evaluated in the cytoplasmic extracts by western blot. Monocytes from normal donors were activated with LPS (1 μg/ml) in combination with IFNα or ethanol overnight and TNFα, IL-6, and IL-12 were measured in the supernatants. Results: In HepG2 cells, both IFNα and acute alcohol treatment induced NF-κB activation and augmented TNFα-induced NF-κB binding. Pretreatment of HepG2 cells with IFNα resulted in the highest levels of NF-κB activation in response to TNFα or TNFα plus ethanol stimulation. Supershift experiments confirmed that the NF-κB dimer induced by TNFα and its combination with IFNα or ethanol contains RelA (p65) and involves rapid degradation of IκBα. Experiments using the proteasome inhibitor, MG132, revealed that augmentation of NF-κB by ethanol and IFNα is mediated via the proteasome pathway. We show that in normal monocytes, IFNα augments LPS-induced production of the inflammatory cytokines TNFα, IL-6, and IL-12 ( p < 0.06) without further modulation by acute alcohol treatment. Conclusions: These results suggest that IFNα can increase HepG2 cell sensitivity to TNFα and ethanol-mediated activation. Augmentation of monocyte inflammatory cytokines, particularly of IL-12 production, by IFNα could be a key element of the antiviral response in chronic HCV. These results support the hypothesis that the therapeutic benefits of IFNα likely involve activation of both immune and parenchymal cells in the liver. [ABSTRACT FROM AUTHOR]

The immunomodulatory capacity of acute, moderate alcohol consumption was investigated in this study in nonalcoholic volunteers after 2 ml of vodka/kg body weight of alcohol consumption. There was a significant, transient increase in interleukin-12 and interferon-γ (IFN γ) levels in whole blood samples collected 4 hr after alcohol consumption in response to an ex vivo bacterial challenge with lipopolysaccharide ( p < 0.02). However, decreased IFN γ levels were produced by mononuclear cells collected later after alcohol consumption (16 hr), suggesting that acute alcohol consumption has a biphasic effect on IFN γ inducibility. Furthermore, isolated blood monocytes collected 16 hr after alcohol consumption showed significantly decreased IL-1β production in response to subsequent bacterial stimulation, implying that in vivo alcohol consumption affects monocyte-derived inflammatory cytokine production. These results demonstrate that even acute, moderate alcohol consumption has a modulating capacity on immune functions that may contribute to decreased immunity and host defense. [ABSTRACT FROM AUTHOR]

We and others have previously shown that even acute ethanol exposure has the capacity to modulate immune functions, particularly monocyte functions. Herein, we tested the hypothesis that acute ethanol treatment inhibits inflammatory, while increasing inhibitory cytokine production in human blood monocytes that, in tum, could contribute to the overall immune abnormalities seen after alcohol use. Our data show that in vitro treatment of blood monocytes with a physiologically relevant dose of alcohol (W mM) results in significantly decreased induction of tumor necrosis factor-α (TNFα) and interleukin (IL)-lβ by bacterial stimulation of either Gram-positive [staphylococcal enterotoxin B (SEB), 1 μg/ml of SEB] or Gram-negative [lipopolysaccharide (LPS), 1 μg/ml of LPS] origin both at the protein and mRNA levels. In contrast, acute ethanol treatment induces monocyte production of mediators with immunoinhibitory potential, including transforming growth factor-β and IL-10. We further show that ethanol not only induces monocyte/macrophage (Mø) IL-10 and transforming growth factor-β, but even augments bacterial (both LPS and SEB) stimulation-induced production of both of these cytokines. IL-10 is a potent inhibitor of Mø TNFα production. We found that ethanol-induced elevation in Mø IL-10 levels contributes to the decreased Mø TNFα production to bacterial challenge in eth-anol-exposed Mø. However, mRNA levels for TNFα are downregu-lated as early as 1.5 hr after ethanol treatment, suggesting that ethanol likely has an IL-10 independent, direct effect on early signaling events of TNF α induction. [ABSTRACT FROM AUTHOR]

Retinoic acid (RA), a metabolic product of vitamin A, has been shown to affect a variety of immune functions, including monocytes. Monocyte functions and mediator production are also modulated by ethanol exposure. This study demonstrates that therapeutic doses of RA (0.1-10 μM) significantly increase transforming growth factor- β (TGFβ) production both in THP-1, human myelomonocytic cells, and in human peripheral blood monocytes. We have previously reported TGFB induction by ethanol in human M ø. Combination of RA stimulation with acute in vitro ethanol treatment, however, resulted in significantly lower Mø TGFβ production than TGFB levels induced by RA alone ( p < 0.003). Down-regulation of M ø TGFβ production by ethanol was tested at the concentration range of 25-150 mM and occurred both at high and low RA concentrations (10-0.1μM). In contrast to its inhibitory effect on RA-induced M ø TGFβ production, ethanol augmented TGFB production induced by mura-my1 dipeptide (20 μg/ml), suggesting that ethanol can either up- or down-regulate Mø TGFB production, depending on the costimulatory factors. RA also induced a moderate increase in Mø tumor necrosis factor-α (TNFα) production, which was down-regulated by ethanol both at the level of secreted and cell-associated TNFα. In addition to regulation of cytokine production, both RA and ethanol decreased expression of CD4 on THP-1 cells. The degree of inhibition of CD4 expression by RA was more significant than by ethanol, but RA-induced decrease in CD4 expression was not significantly affected by the combined stimulation with ethanol. These results provide further evidence for the immunoregulatory potential of nutritional and dietary factors, such as RA and ethanol, on the immune functions of human monocytes. [ABSTRACT FROM AUTHOR]