BACKGROUND Chronic alcohol abuse is a comorbid variable of Acute Respiratory Distress Syndrome (ARDS). Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Nox) are the main source of reactive oxygen species (ROS) in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2 and Nox4 expression. METHODS AMs were isolated from male C57BL/6J mice, aged 8-10 weeks, which were treated ± ethanol in drinking water (20% w/v, 12 weeks). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox siRNAs (20-35 nM), prior to ethanol exposure. Human AMs were isolated from alcoholic and control patients’ bronchoalveolar lavage fluid. Nox mRNA levels (qRT-PCR), protein levels (western blot and immunostaining), oxidative stress (DCFH-DA and Amplex Red analysis), and phagocytosis (S. aureus internalization) were measured. RESULTS Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox siRNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2 and Nox4 protein levels were augmented in human AMs from alcoholics compared with controls. CONCLUSIONS Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.
Chronic alcohol consumption leads to inflammation and cirrhosis of the liver. In this study, we observed that liver sinusoidal endothelial cells (LSEC) derived from ethanol-fed rats showed several fold increases in the mRNA expression of endothelin-1 (ET-1), hypoxia-inducible factor-1α (HIF-1α), and inflammatory cytochemokines compared with control rat LSEC. We also observed the same results in acute ethanol-treated LSEC from control rats and human dermal microvascular endothelial cells. Ethanol-mediated ET-1 expression involved NADPH oxidase and HIF-1α activation. Furthermore, ethanol increased the expression of the ET-1 cognate receptor ET-BR in Kupffer cells and THP-1 monocytic cells, which also involved HIF-1α activation. Promoter analysis and chromatin immunoprecipitation showed that hypoxia response element sites in the proximal promoter of ET-1 and ET-BR were required for the binding of HIF-1α to up-regulate their expression. We showed that microRNAs, miR-199 among several microRNAs, attenuated HIF-1α and ET-1 expression, while anti-miR-199 reversed the effects, suggesting that ethanol-induced miR-199 down-regulation may contribute to augmented HIF-1α and ET-1 expression. Our studies, for the first time to our knowledge, show that ethanol-mediated ET-1 and ET-BR expression involve HIF-1α, independent of hypoxia. Additionally, ethanol-induced ET-1 expression in rat LSEC is regulated by miR-199, while in human endothelial cells, ET-1 expression is regulated by miR-199 and miR-155, indicating that these microRNAs may function as novel negative regulators to control ET-1 transcription and, thus, homeostatic levels of ET-1 to maintain microcirculatory tone.
Peroxisome proliferator-activated receptor (PPAR) g is critical for alveolar macrophage (AM) function. Chronic alcohol abuse causes AM phagocytic dysfunction and susceptibility to respiratory infections by stimulating nicotinamide adenine dinucleotide oxidases (Nox), transforming growth factor-b 1 , and oxidative stress in the AM. Because PPARg inhibits Nox expression, we hypothesized that alcohol reduces PPARg, stimulating AM dysfunction. AMs were examined from: (1) patients with alcoholism or control patients; (2) a mouse model of chronic ethanol consumption; (3) PPARg knockout mice; or (4) MH-S cells exposed to ethanol in vitro. Alcohol reduced AM PPARg levels and increased Nox1, -2, and -4, transforming growth factor-b 1 , oxidative stress, and phagocytic dysfunction. Genetic loss of PPARg recapitulated, whereas stimulating PPARg activity attenuated alcohol-mediated alterations in gene expression and phagocytic function, supporting the importance of PPARg in alcohol-induced AM derangements. Similarly, PPARg activation in vivo reduced alcohol-mediated impairments in lung bacterial clearance. Alcohol increased levels of microRNA-130a/-301a, which bind to the PPARg 39 untranslated region to reduce PPARg expression. MicroRNA-130a/-301a inhibition attenuated alcohol-mediated PPARg reductions and derangements in AM gene expression and function. Alcohol-induced Toll-like receptor 4 endocytosis was reversed by PPARg activation. These findings demonstrate that targeting PPARg provides a novel therapeutic approach for mitigating alcohol-induced AM derangements and susceptibility to lung infection.
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