Alcohol abuse is associated with immunosuppressive and infectious sequelae. Particularly, alcoholics are more susceptible to pulmonary infections. In this report, gene transcriptional profiles of primary human airway epithelial cells exposed to varying doses of alcohol (0, 50, and 100 mM) were obtained. Comparison of gene transcription levels in 0 mM alcohol treatments with those in 50 mM alcohol treatments resulted in 2 genes being upregulated and 16 genes downregulated by at least 2-fold. Moreover, 0 mM and 100 mM alcohol exposure led to the upregulation of 14 genes and downregulation of 157 genes. Among the upregulated genes, glucocorticoid-induced leucine zipper (GILZ) responded to alcohol in a dose-dependent manner. Moreover, GILZ protein levels also correlated with this transcriptional pattern. Lentiviral expression of GILZ small interfering RNA in human airway epithelial cells diminished the alcohol-induced upregulation, confirming that GILZ is indeed an alcohol-responsive gene. Gene silencing of GILZ in A549 cells resulted in secretion of significantly higher amounts of inflammatory cytokines in response to IL-1β stimulation. The GILZ-silenced cells were more resistant to alcohol-mediated suppression of cytokine secretion. Further data demonstrated that the glucocorticoid receptor is involved in the regulation of GILZ by alcohol. Because GILZ is a key glucocorticoid-responsive factor mediating the anti-inflammatory and immunosuppressive actions of steroids, we propose that similar signaling pathways may play a role in the anti-inflammatory and immunosuppressive effects of alcohol.

Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM) for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (I SC ) in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A 2B adenosine receptor (A 2B AR), largely abolished the adenosine-stimulated chloride transport, suggesting that A 2B AR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A 2B AR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

Alcohol abuse has been associated with increased susceptibility to pulmonary infection. It is not fully defined how alcohol contributes to the host defense compromise. Here primary human airway epithelial cells were cultured at an air-liquid interface to form a differentiated and polarized epithelium. This unique culture model allowed us to closely mimic lung infection in the context of alcohol abuse by basolateral alcohol exposure and apical live bacterial challenge. Application of clinically relevant concentrations of alcohol for 24 hours did not significantly alter epithelial integrity or barrier function. When apically challenged with viable Klebsiella pneumoniae, the cultured epithelia had an enhanced tightness which was unaffected by alcohol. Further, alcohol enhanced apical bacterial growth, but not bacterial binding to the cells. The cultured epithelium in the absence of any treatment or stimulation had a base-level IL-6 and IL-8 secretion. Apical bacterial challenge significantly elevated the basolateral secretion of inflammatory cytokines including IL-2, IL-4, IL-6, IL-8, IFN-γ, GM-CSF, and TNF-α. However, alcohol suppressed the observed cytokine burst in response to infection. Addition of adenosine receptor agonists negated the suppression of IL-6 and TNF-α. Thus, acute alcohol alters the epithelial cytokine response to infection, which can be partially mitigated by adenosine receptor agonists.

No abstract

We previously reported the presence of Angiotensin Converting Enzyme type 2 (ACE2) in brain regions involved in cardiovascular regulation. Here we test the role of central ACE2 on oxidative stress and autonomic function. ACE2 knockout (KO) mice (n=3/group) were infected with a hACE2 or control (eGFP) adenovirus (2×10e6 pfu, 200 nL) in the paraventricular nucleus (PVN) 3d before AngII infusion (osmotic pump: 600 ng/kg/min sc). After 4d infusion, mice were injected with dihydroethidium (DHE) intracardially and staining measured in brain regions (Image J, arbitrary units). Baseline blood pressure (BP) (telemetry: 112±7 vs. 111±3 mmHg) and DHE staining in the brain were not different between ACE2 KO and wildtypes. Post infusion, AngII levels in the hypothalamus were significantly higher in KO vs. wildtype mice (RIA: 2.9±0.3 vs. 2.0±0.2 fmol/mg, n=5, P<0.05) and DHE fluorescence in KO (eGFP‐infected) dramatically increased (P<0.01) vs. baseline in the PVN (194±12 vs. 101±3), NTS (52±2 vs. 20±2) and RVLM (207±22 vs. 77±12). This increase was prevented in Ad‐hACE2‐infected KO in the PVN (114±8, P<0.01) as well as in the NTS (22±2, P<0.01) and RVLM (88±8, P<0.01). Moreover, ACE2 gene therapy decreased the LF/HF ratio (BP variability) in AngII‐infused KO (5±1 vs. 9±3). These data support the protective role of ACE2 in the central regulation of autonomic function by reducing oxidative stress. (NIH NS052479 and P20RR018766)