Background Alcohol abuse has long-term deleterious effects on the immune system, and results in a depletion and loss of function of CD4+ T lymphocytes, which regulate both innate and adaptive immunity. T-lymphocyte activation via T-cell receptor (TCR) involves the lipid raft colocalization and aggregation of proteins into the immunological signalosome, which triggers a signaling cascade resulting in the production of interleukin-2 (IL-2). IL-2 regulates the proliferation and clonal expansion of activated T cells and is essential for an effective immune response. The present work examines the mechanisms underlying ethanol-induced dysfunction of CD4+ T lymphocytes based on the hypothesis that ethanol downregulates lipid raft-mediated TCR signal transduction and resultant IL-2 production. Methods Primary or cultured human T lymphocytes were exposed to ethanol for 24 hours prior to stimulation with anti-CD3/anti-CD28 antibodies or phytohemagglutinin. Effects of ethanol exposure on TCR-signaling (including activation of Lck, ZAP70, LAT, and PLCγ1) and IL-2 gene expression were examined. Results Exposure of both primary and cultured human CD4+ T lymphocytes to physiologically relevant concentrations of ethanol leads to down-regulation of IL-2 mRNA and protein via inhibition of DNA-binding activity of NFAT, the essential transcription factor for IL-2. Ethanol decreases tyrosine phosphorylation and activation of upstream signaling proteins PLCγ1, LAT, ZAP70, and Lck. These effects are prevented by inhibition of metabolism of ethanol. Sucrose density gradient fractionation and confocal microscopy revealed that ethanol inhibited essential upstream lipid raft-mediated TCR-dependent signaling events, namely colocalization of Lck, ZAP70, LAT, and PLCγ1 with plasma membrane lipid rafts. Conclusions Overall, our data demonstrate that ethanol inhibits lipid raft-mediated TCR-signaling in CD4+ T lymphocytes, resulting in suppression of IL-2 production. These findings may represent a novel mechanism underlying alcohol abuse-associated immune suppression and may be particularly relevant in diseases such as HIV/AIDS and hepatitis C virus infection where alcohol abuse is a known comorbidity.

Activation-induced Fas Ligand (FasL) mRNA expression in CD4+ T cells is mainly controlled at transcriptional initiation. To elucidate the epigenetic mechanisms regulating physiologic and pathologic FasL transcription, TCR-stimulation responsive promoter histone modifications in normal and alcohol-exposed primary human CD4+ T cells were examined. TCR stimulation of normal and alcohol-exposed cells led to discernible changes in promoter histone H3 lysine trimethylation, documented by an increase in the levels of transcriptionally permissive H3K4Me3 and a concomitant decrease in the repressive H3K9Me3. Moreover, acetylation of H3K9 (H3K9Ac), a critical feature of the active promoter state which is opposed by H3K9Me3, was significantly increased and was essentially mediated by the p300-histone acetyltransferase (p300-HAT). Notably, the degree of these coordinated histone modifications and subsequent recruitment of transcription factors and RNA polymerase II (RNA Pol II) was significantly enhanced in alcohol exposed CD4+ T cells and was commensurate with the pathologic increase in the levels of FasL mRNA. The clinical relevance of these findings is further supported by CD4+ T cells obtained from individuals with a history of heavy alcohol consumption that demonstrate significantly greater p300-dependent H3K9 acetylation and FasL expression. Overall, these data show that in human CD4+ T cells, TCR stimulation induces a distinct promoter histone profile involving a coordinated crosstalk between H3K4 and H3K9 methylation and acetylation that dictates the transcriptional activation of FasL under physiologic as well as pathologic conditions of alcohol exposure.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and can lead to hepatocellular carcinoma and end-stage liver disease. The current FDA-approved treatment for HCV (pegylated interferon-alpha (IFNα) with ribavirin) is effective only in about 50% of patients. Epidemiological evidence suggests that obesity, alcohol, smoking and environmental pollutants may contribute to resistance to IFNα therapy in HCV. Acrolein, a ubiquitous environmental pollutant and major component of cigarette smoke, is also generated endogenously by cellular metabolism and lipid peroxidation. This study examines the effects of acrolein on (i) IFNα-mediated signaling and antiviral gene expression in cultured and primary human hepatocytes, and (ii) HCV replication in an HCV-replicon system. Our data demonstrate that non-toxic concentrations of acrolein significantly inhibited IFNα-induced tyrosine phosphorylation of both cytoplasmic and nuclear STAT1 and STAT2, without altering the total levels. Also, acrolein down-regulated IFNα stimulated gene transcription, resulting in reduced expression of antiviral genes. Importantly, acrolein abolished the IFNα mediated downregulation of HCV viral expression in the HCV-replicon system. This study defines mechanisms involved in resistance to IFNα and identifies the pathogenic role of acrolein, and potentially other environmental pollutants, in suppressing IFNα antiviral activity, and establishes their adverse impact on HCV therapy.

Alcohol abuse has long‐term deleterious effects on the immune system, and results in reduced function of CD4+T lymphocytes, which regulate both innate and adaptive immunity. T lymphocyte activation via T‐cell receptor (TCR) involves the plasma membrane lipid raft‐dependent aggregation of proteins into the immunological signalosome, which in turn, triggers a signaling cascade resulting in interleukin‐2 (IL‐2) production. IL‐2 is a critical cytokine that regulates the proliferation/clonal expansion of activated T‐cells, and is essential for an effective immune response.The present work examines the mechanisms underlying ethanol‐induced dysfunction of CD4+T lymphocytes, and demonstrates for the first time, that physiologically relevant concentrations of ethanol inhibit lipid raft‐dependent TCR signaling and decrease production of IL‐2 in both primary and cultured human CD4+T lymphocytes. Specifically, ethanol substantially down‐regulates lipid raft colocalization and activation/phosphorylation of Lck, ZAP70 and LAT, resulting in a marked decrease in DNA‐binding activity of NFAT and IL‐2 mRNA and protein. Importantly, inhibition of ethanol metabolism prevented these inhibitory effects. Overall, our findings have clinical relevance in alcohol abuse‐associated immune dysfunction, especially in conditions such as HIV and HCV infection where alcohol abuse is a known comorbidity.

No abstract