Ethanol Upregulates Glucocorticoid-Induced Leucine Zipper Expression and Modulates Cellular Inflammatory Responses in Lung Epithelial Cells

According to the National Epidemiologic Survey on Alcohol and Related Conditions, greater than one third of the population in the United States aged 18 and older consumes alcohol. Ethanol, the active component in all alcoholic beverages, produces a wide variety of behavioral and physiological effects in humans (1, 2). Alcohol has long been known as an inflammation-inhibitory and immunosuppressive agent. Abuse of this substance is clearly linked to organ and tissue damage (3), host susceptibility to infectious diseases (4, 5), and specific defects in innate and adaptive immunity (6). However, the molecular mechanisms for these effects of alcohol have not been completely defined.

Glucocorticoid (GC) hormones are potent modulators of immune responses and inflammatory processes. GCs are the most commonly used anti-inflammatory and immunosuppressive drugs in the treatment of rheumatic and other inflammatory diseases. Cellular effects of GCs are mediated through the glucocorticoid receptor (GR), which regulates the expression of GC-responsive genes (7, 8). One of the prominent GC-responsive genes is the glucocorticoid-induced leucine zipper (GILZ) (9, 10). GILZ controls T cell activation and development, and inhibits the activities of the key inflammatory signaling mediators NF-κb and AP-1 (9, 11, 12). When stimulated with GCs, macrophages synthesize significant amounts of GILZ, which subsequently attenuates macrophage inflammatory responses (13). Moreover, GILZ gene expression is downregulated in macrophages from inflammatory lesions of delayed-type hypersensitivity reactions, and persists in tumor-infiltrating macrophages from Burkitt lymphoma (13). GILZ mediates the anti-inflammatory effects of GCs via suppressing the activation of NF-κb in human airway epithelial cells (14) and redirects the maturation of dendritic cells, preventing Ag-specific T lymphocyte responses (15). These data strongly suggest that GILZ plays a key role in inflammatory inhibition and immunosuppression. GILZ has several isoforms owing to alternative RNA splicing: GILZ-1 (137 aa), GILZ-2 (201 aa), GILZ-3 (43 aa), and GILZ-4 (80 aa) (16). These isoforms were found to have striking functional differences in various types of cells. In the current study, we report that human airway epithelial cells respond to alcohol by modulating their transcriptome profiles. Among the identified alcohol-responsive genes, GILZ, with an m.w. of ∼17 KDa, which is correlated to the GILZ1 in the previous publications (14, 16), is markedly upregulated. Further experimental data suggest that GILZ upregulation may serve as a key factor for alcohol to modulate inflammatory responses by sharing the same signaling pathway with GCs.

Primary NHBE cells (Lot #7F3477; Lonza Walkersville, Walkersville, MA), A549 cells, BEAS-2B, and HUVEC-C cells (American Type Culture Collection) were cultured according to the recommended protocols by the manufacturers. The air–liquid interface culture protocol was modified from the previous publication (17). Briefly, 5 × 10⁵ NHBE cells were seeded on collagen-coated Millicell-PCF membrane inserts (Millipore, Billerica, MA). After the cells were cultured in submersion for 5 d, the apical medium was aspirated off and the cells were grown at the air–liquid interface for 2 more wk. This air–liquid culture condition closely mimics the in vivo airways, and the culture was previously shown to be fully differentiated, pseudostratified airway epithelia (18).

The air–liquid interface cultures were exposed to alcohol by adding ethanol (200 proof; AAPER Alcohol and Chemical Co., Shelbyville, KY) at varying doses to the basal medium. All other submerged cultures were treated with alcohol by replacing with ethanol-containing culture media. The alcohol exposure time and concentration are indicated in the individual experiment. All the cultures were kept in 37°C, 5% CO₂ incubators that had been presaturated with the specified ethanol concentration. Whenever indicated, dexamethasone (Sigma-Aldrich, St. Louis, MO) at 1 μM or RU-486 (mifepristone) at 10 μM (Sigma-Aldrich) was applied.

Total RNAs were extracted from three different lots of NHBE cells that had been cultured at the air–liquid interface and treated with different concentrations of ethanol for 24 h, using the RNeasy Mini Kit (Qiagen, Valencia, CA). Procedures for cDNA synthesis, labeling, and hybridization were carried out as described at www.affymetrix.com/support/technical/manual/expression_manual.affx (Affymetrix, Redwood City, CA). All experiments were performed using human genome U133 plus 2.0 Genechips as described at www.affymetrix.com/support/technical/byproduct.affx?product = hg-u133-plus. The CEL data file from each array was imported into Genespring GX 7.3 (Agilent Technologies, Santa Clara, CA) and preprocessed with the robust multichip average method, and per gene normalization was applied using the median values of the no ethanol control samples. The detection call metrics from the CHP files were used to filter out transcripts found to be absent in all nine samples. In addition, transcripts that were not within a SD of 1.4 and exhibiting <1.5-fold change were also removed from further analysis. To identify upregulated and downregulated transcripts, genes were filtered using the volcano plot option with the limit set at p < 0.01 and at least a 2.0-fold change. The parametric test, with variance not equal, was applied without multiple test correction. The gene profile data have been deposited to Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/; access number GSE12253).

Western blot assays were performed according to the published procedure, with modifications (19). Cells were washed twice with ice-cold PBS followed by lysis in Nonidet P-40 lysis buffer (50 mM Tris/HCl, pH 8.0; 0.5% Nonidet P-40; 150 mM NaCl; 0.1 mM EDTA; 10 mM NaF; 1 mM PMSF; and protease inhibitors mixture [Roche, Basel, Switzerland]). Samples were passed three times sequentially through 25G and 27G needles and centrifuged at 14,000 rpm for 20 min at 4°C. The supernatants were collected, and the protein concentrations were determined using a BCA Assay (Pierce, Rockford, IL). An equal amount of protein, as indicated, was loaded per sample for electrophoresis. The resolved proteins were transferred onto a nitrocellulose membrane and the primary Ab—rabbit anti-GILZ (FL-134) (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-Actin (I-19) (1:1000; Santa Cruz Biotechnology)—was used. The secondary Ab was goat anti-Rabbit–IgG conjugated HRP (1:20,000; Bio-Rad, Hercules, CA). As needed for reprobing, the membranes were stripped at 50°C for 30 min in stripping buffer (62.5 mM Tris/HCl, pH 6.8; 2% SDS; and 100 mM 2-mercaptoethanol). For quantification, autoradiograms with an unsaturated exposure were scanned using Alpha Imager EC (Alpha Innotech, San Leandro, CA) and analyzed with National Institutes of Health ImageJ software (National Institutes of Health, Bethesda, MD). Signal intensity was adjusted for the background of each blot. Data are presented as the ratio of signal intensity of the test protein to that of β-actin loading control.

The short hairpin structure for GILZ gene silencing was constructed as follows. For GILZ silence (siGILZ), the two complementary oligos were: 5′-AACAGCTTCACCTGACAACGACTTCCTGTCATCGTTGTCAGGTGAAGCTGTTTTTTTT-3′ and 5′-TCGAAAAAAAAACAGCTTCACCTGACAACGATGACAGGAAGTCGT TGTCAGGTGAAGCTGTTGGCC-3′. For the scrambled RNA control (siCNTL), the two complementary oligos were 5′-CATAACGAGCGGAAGAACGCTTCCTGTCACGTTCTTCCGCTCGTTATGTTTTTT-3′ and 5′-TCGAAAAAAACATAACGAGCGGAAGAACGTGACAGGAAGCGTTCTTCCGCTCGTTATGGGCC-3′. The ApaI and XhoI restriction enzyme sites were engineered at the ends of each oligo. The hairpin loops are shown in bold. The complementary oligo pairs were annealed and ligated into a pSILENCER-1.0 vector (Ambion, Austin, TX) at the ApaI and XhoI sites. The expression cassettes containing the mouse U6 promoter and siGILZ or siCNTL were cloned into the HIV-based lentiviral vector system to generate Lenti-U6-siGILZ-CMV-EGFP and Lenti-U6-siCNTL-CMV-EGFP transgene plasmids. Each of the transgene plasmids has two separate expression cassettes: the U6-siGILZ or U6-siCNTL cassette and the CMV-EGFP cassette. For lentiviral vector production, HEK 293T cells were transfected, via the calcium phosphate precipitation method, with triple plasmids: 1) the envelope plasmid pLTR-G, 2) the packaging plasmid pCD/NL-BH*ΔΔΔ, and 3) one of the transgene plasmids(20). Forty-eight hours later, the viral vector-containing culture media were collected and vectors were concentrated by ultracentrifugation.

The EGFP-GRα fusion expression vector was constructed from the cDNA of NHBE cells. GRα was amplified by RT-PCR, using the forward primer containing an Xho I site (5′-CCGCTCGAGCATGGACTCCAAAGAATCATT-3′) and the reverse primer containing a BamH1 site (5′-CCGGGATCCTCACTTTTGATGAAACAGAAG-3′) in a high-fidelity PCR supermix (In-vitrogen, Carlsbad, CA). The resulting PCR product was digested with Xho1 and BamH1 and cloned into the expression vector pEGFP-C2 (Clontech, Mountain View, CA). The YFP-GRβ was graciously provided by Dr. Cidlowski at the National Institutes of Health (21). Owing to the ease and high efficiency of transfection by calcium phosphate precipitation, HEK-293T cells were selected to study the effects of dexamethasone and alcohol on translocation of the two fusion constructs. Because of the emission spectrum overlap, the yellow fluorescent protein (YFP) fusion protein can be observed using the same filter combination as enhanced green fluorescence protein (EGFP).

Human distal lung epithelial A549 cells were transduced, respectively, with Lenti-pU6-siGILZ-pCMV-EGFP or Lenti-pU6-siCNTL-pCMV-EGFP at a multiplicity of infection of 5. The cells were then FACS-sorted to a pure population and referred to as A549-siGILZ and A549-siCNTL. GILZ gene silencing was confirmed by Western blotting. A549-siGILZ and A549-siCNTL were plated at a density of 5 × 10⁵ cells per well in a 6-well dish. These cells were stimulated with 10 ng/ml IL-1β (Calbiochem, San Diego, CA) for 3 h. For the next 24 h, the cells were continually exposed to IL-1β in the presence and absence of 50 mM alcohol. Then, 300 μl media from each well was collected and centrifuged at 14,000 rpm for 20 min at 4°C. The supernatants were analyzed for inflammatory cytokine levels, using the human 8-Plex Cytokine Assay (Bio-Rad) according to the protocol recommended by the manufacturer. The eight human cytokines examined were IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-γ, and TNF-α. For ELISA measurement of IL-6, we used the commercial human IL-6 Quantikine Kit (R&D Systems, Minneapolis, MN) and the recommended protocol.

For statistical analysis in Fig. 4B, Student t tests were conducted as two-tailed pairwise comparisons between the groups. Results are expressed as means ± S.D. A probability level ≤0.05 was considered significant.

To understand the cellular responses of human airway epithelia to alcohol, we first identified those genes whose expressions were significantly altered by alcohol exposure. Well-differentiated NHBE, cultured at an air–liquid interface, were treated with varied doses of alcohol (0, 50, and 100 mM) for 24 h. The samples were then subjected to whole-genome high-density microarray analyses. The obtained data have been deposited to Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/; access number GSE12253). Comparison between 0 mM and 50 mM alcohol treatments resulted in only 2 genes being significantly upregulated and 16 genes significantly downregulated by the 2-fold criterion in our system (Fig. 1A, 1B). The two upregulated genes were 1) TSC22 domain family member 3 or GILZ and 2) a putative gene (human hypothetical protein 644242) with unknown function. Treatment with 100 mM alcohol upregulated 14 genes and downregulated 157 genes by at least 2-fold. These upregulated genes are specifically displayed (Fig. 1B). The profile of genes that showed a dose-dependent increase by at least 1.5-fold with 50 mM and 100 mM alcohol treatments is shown in Table I.

Among the ethanol-upregulated genes, GILZ was elevated by greater than 2-fold at 50 mM and 3-fold at 100 mM alcohol. The alcohol dose-dependent expression pattern of GILZ suggested that GILZ was an alcohol responsive gene and worthy of further investigation. To test whether the levels of GILZ mRNA correlated with the levels of GILZ protein, we performed immunoblot assays using a specific Ab against GILZ. The results showed a clear dose-dependent effect of alcohol on the upregulation of GILZ protein with a m.w. of ∼17 kDa (Fig. 2A). Based on the size of the protein, we judged the detected GILZ to be GILZ1, as documented in previous publications (14, 16). In contrast, the expression level of the internal actin control remained constant.

To test whether alcohol could induce GILZ upregulation in other cell types, human distal lung epithelial A549 cells and HUVECs were examined. Prior to immunoblotting, all cells were cultured in the presence or absence of 100 mM alcohol for 24 h. The results showed that GILZ was similarly upregulated (Fig. 2B) in these cells as well. Thus, alcohol induces GILZ expression not only in primary and immortalized pulmonary epithelial cells but also in primary vascular endothelial cells.

To determine whether GILZ responds to physiologically relevant alcohol concentration, we selected low levels of alcohol (0, 25, and 50 mM) to treat, for 24 h, various pulmonary epithelial cells: NHBE, A549, and BEAS-2B. GILZ expression was similarly assessed by immunoblot assays followed by densitometry analyses. As shown, the level of GILZ protein was increased by alcohol in the three tested cells (Fig. 2C–E), indicating a clear upregulation of GILZ by physiologically encountered concentrations of alcohol.

If alcohol is the causative agent for the induction of GILZ, removal of alcohol should reverse the effect. A549 cells were treated with 100 mM alcohol for 24 h, followed by culturing in alcohol-free media. Cell samples were collected at various time points after alcohol withdrawal, as indicated (0, 4, 8, 18, 24, and 42 h). GILZ expression was measured by immunoblot. As shown, A549 cells expressed little detectable GILZ without alcohol stimulation (Fig. 2F). After a 24 h exposure to 100 mM alcohol, the cells expressed a significant level of GILZ. However, ∼8 h after alcohol withdrawal, GILZ expression decreased to prealcohol treatment levels. These data suggest that GILZ responsiveness to alcohol is reversible after alcohol withdrawal. Moreover, these data also suggest that alcohol’s effect on GILZ is not caused by ethanol-induced cytotoxicity.

To achieve specific gene silencing of GILZ, we employed the small interfering RNA (siRNA) approach by construction of the following two lentiviral vectors (Fig. 3A): 1) Lenti-pU6-siGILZ-pCMV-EGFP and 2) Lenti-pU6-siCNTL-pCMV-EGFP. In both vectors, the mouse U6 promoter drives the expression of either siGILZ or a small siCNTL. In addition, in a separate expression cassette, the CMV promoter drives the expression of EGFP, which serves as an indicator for vector transduction. NHBE cells cultured in submersion were transduced with each of the viral vectors at an multiplicity of infection of 10. As seen (Fig. 3B–E), all the cells expressed EGFP, suggesting that cells were effectively transduced by the vectors and potentially express the corresponding siRNAs. Immunoblot assays were performed to assess the efficacy of GILZ knockdown and the ethanol effect on GILZ in the cells. Total cellular proteins (15 μg per lane) were loaded and probed with the specific Ab against GILZ or actin. GILZ expression in the cells transduced with Lenti-pU6-siGILZ-pCMV-EGFP was not detectable by immunoblot, and alcohol exposure failed to upregulate GILZ expression in these cells (Fig. 3F). In contrast, A549 cells receiving the control vector responded to alcohol normally by upregulation of GILZ expression. These data strongly suggest that GILZ is a true alcohol-responsive gene.

To delineate the role of GILZ in cellular responses to inflammatory stimuli and alcohol-induced immunosuppression, two cell lines derived from A549 cells with either permanent GILZ knockdown (A549-siGILZ) or mock siRNA control (A549-siCNTL) were generated. These cells were stimulated by adding IL-1β (10 ng/ml) 3 h prior to treatment, with or without 50 mM alcohol for 24 h. The secretion of eight inflammatory cytokines (IL-2, IL-4, IL-6, GM-CSF, IFN-γ, TNF-α, IL-8, and IL-10) was screened by Bio-Plex Cytokine Assays. Without IL-1β stimulation, both cells produced appreciable basal levels of IL-6 and IL-8. However, the other measured cytokines were produced at a minimal level and near the threshold of detection (Table II). With IL-1β stimulation, six of eight cytokines (IL-2, IL-4, IL-6, GM-CSF, IFN- γ, and TNF-α) were increased by 4.3-, 4.9-, 3.4-, 5.1-, 5.1-, and 5.5-fold, respectively, in the control A549-siCNTL cells (Fig. 4A). In contrast to control cells, these 6 cytokines showed a significantly greater fold increase in GILZ-silenced A549 cells after IL-1β stimulation (5.3-, 7.3-, 8.1-, 9.1-, 9.1-, and 12.2-fold for each cytokine, respectively). Secretion of IL-8 and IL-10 was not affected by IL-1β stimulation in these cells (data not shown). Ethanol, applied 3 h after IL-1β stimulation, significantly reduced the stimulated secretion of IL-2, IL-4, IL-6, GM-CSF, IFN- γ, and TNF-α secreted by control A549 cells. In contrast, ethanol treatment had a far lesser suppressive effect on the stimulated secretion of these cytokines in GILZ knockdown cells (Fig. 4A). Among these cytokines, the cytokine producing the highest amounts and most affected by ethanol was IL-6. Based on this finding, we confirmed our Bio-Plex findings by assaying IL-6 by ELISA. As shown in Fig. 4B, A549-siGILZ responded to IL-1β stimulation by increasing IL-6 secretion by a much greater magnitude than the control A549-siCNTL cells. Again, GILZ-silenced cells were significantly more resistant to the suppressive effects of ethanol.

Because GILZ is known to be upregulated by GCs via binding to GR, we hypothesized that alcohol might be acting through the same signaling pathway as GCs. To test this hypothesis, we first used the GR-specific antagonist RU-486. Primary human airway epithelial cells were cultured in 100 mM alcohol medium in the presence or absence of RU-486 (10 μM). Primary human airway epithelial cells had a basal expression of GILZ, and alcohol again stimulated GILZ expression in these cells (Fig. 5A). RU-486 was able to substantially suppress the alcohol-stimulated GILZ expression. This incomplete suppression was previously observed in RU-486 inhibition of GILZ expression induced with dexamethasone (14, 22) owing to the dual activities of RU-486 as a potent GC antagonist and a partial GC agonist. These results suggest that GR might be involved in the alcohol-induced GILZ expression. Because GR is a ligand-dependent transcription factor, it must be translocated to the cell nucleus to regulate expression of GC-targeted genes. Thus, GR localization can be used to judge whether alcohol stimulates GILZ expression through GR. Toward this end, we constructed an EGFP-GRα fusion protein expression vector, in which the EGFP gene was fused, in-frame, with GRα at its N terminus. In addition, a vector expressing the YFP-GRβ fusion protein was obtained to assess GRβ localization (20). HEK-293T cells were transfected with each of the following three vectors: 1) pEGFP, 2) pEGFP-GRα, and 3) pYFP-GRβ. At 24 h after transfection, the cells were cultured in control medium (Fig. 5B, 5E, 5H) or supplemented with 1 μM dexamethasone (Fig. 5C, 5F, 5I) or 100 mM alcohol (Fig. 5D, 5G, 5J). After dexamethasone stimulation, EGFP-GRα was completely translocated into the cell nuclei (Fig. 5F), and YFP-GRβ partially into the cell nuclei (Fig. 5I). Notably, as with dexamethasone, alcohol exposure resulted in nuclear translocation of GRα and GRβ, suggesting the involvement of GR in alcohol induction of GILZ expression.

Alcohol produces a broad spectrum of effects in the human body. The molecular mechanisms underlying these effects are not fully understood. The current report documents that cells specifically alter their gene transcriptional profile in response to alcohol exposure, suggesting that gene regulation might be a mechanism by which alcohol can act on cells. Accumulated data demonstrate that moderate alcohol intake has been associated with reductions in many adverse health conditions, including coronary artery disease, diabetes, hypertension, congestive heart failure, stroke, arthritis, and dementia (2, 23). Moderate consumption of alcohol is also found to benefit overall survival and quality of life in the elderly (24). However, abuse of this substance leads to detrimental consequences, including immunosuppression and infections. In this report, we have shown that GILZ is one of the genes significantly upregulated by alcohol. Such an upregulation recapitulates the GILZ response to GCs. Clinically, GCs are the most commonly prescribed drugs for management of inflammation. Our finding that alcohol conveys effects similar to those of GCs, although at a lower potency than dexamethasone, predicts that moderate alcohol intake may mediate its beneficial effects through this signaling pathway. Interestingly, previous clinical data demonstrate that alcohol decreases the risk of certain autoimmune inflammatory disorders, such as rheumatoid arthritis (25, 26), and has been associated with improvement in asthma symptoms (27).

It is well documented that alcohol abuse predisposes individuals to infections by bacteria, fungi, and viruses (4–6), and consequently alcohol-abusing patients suffer higher mortality from bacterial pneumonia than do control patient populations (28). Microbial infections elicit many inflammatory pathways in the lung, which is an essential response required to mobilize and orchestrate both innate and adaptive host defenses. Depending on how the host is exposed to alcohol, pulmonary inflammatory responses could vary (29). Our data and previous publications show that acute ethanol exposure inhibits the production of inflammatory cytokines (30–33). However, chronic ethanol exposure has varied effects on inflammatory cytokines. In rats, chronic ethanol ingestion decreases TNF-α production by alveolar macrophages (34) and downregulates GM-CSF receptor expression on the cell surface to suppress inflammation (35). In contrast, chronic ethanol abuse results in enhanced macrophage production of TNF-α from both human PBMCs and mouse Kupffer cells (36, 37). Moreover, alcohol abusers can have elevated inflammatory cytokine responses (29). These multifaceted effects of alcohol on lung inflammatory responses indicate a complex regulation of the process. Our data demonstrated that short-term alcohol exposure induces reversible GILZ upregulation in pulmonary epithelial cells, which may be of relevance to lung inflammatory responses. The effect of chronic alcohol exposure on GILZ expression remains to be determined. Nevertheless, GILZ is a TGF β1-stimulated protein. Chronic alcohol ingestion in rats increases TGF-β1 expression in the lung (38). Given that TGF-β1 is an upstream regulator gene, it is possible that GILZ might be upregulated through this mechanism. Thus, further research is required to determine if GILZ upregulation is a direct effect of alcohol or occurs through an autocrine/paracrine signaling pathway. GILZ overexpression in transgenic mice inhibits Th1 CD4⁺ T cells responses and enhances Th2 responses (39). In airway epithelial cells, GILZ suppresses activation of NF-κb promoter, which attenuates expression of the downstream luciferase gene in response to cytokine induction (14). Therefore, alcohol-induced GILZ upregulation may contribute to the overall modulation of airway inflammatory responses when infections occur.

Our microarray data revealed that many genes are significantly upregulated or downregulated by alcohol exposure. In addition to GILZ, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) also showed a dose-dependent upregulation by alcohol (Table I). PTEN is a major negative regulator of the PI3–kinase pathway and antagonizes PI3 kinase activity (40). This signaling pathway is responsible for cell proliferation and survival. Moreover, T cells with PTEN deficiency contribute to autoimmune diseases (41). Interestingly, a recent publication documented that GILZ is not only regulated by the GC–GR pathway but also regulated by the PI3-kinase/AKT pathway (42). Therefore, PTEN may be another upstream gene that controls GILZ expression in this system. Even though the current report focuses on GILZ, we are aware of the potential importance of other candidate genes. Future investigations are warranted to understand the interplay of the genes and to map the associated pathways and signaling networks.

In this report, we used the Bio-Plex Cytokine Assay to screen for basal and IL-1β–stimulated cytokine responses in the A549-derived cell lines. These cells produced IL-6 and IL-8 at the low nanogram per milliliter level and the other measured cytokines (IL-2, IL-4, GM-CSF, IFN- γ, and TNF-α) at the low picogram per milliliter level. As IL-6 showed the greatest induction, we used conventional ELISA to selectively confirm this result. In the course of this study, we noticed that the two methods had varied detection sensitivities. The Bio-Plex method appeared to be much more sensitive than ELISA, probably owing to the differences of the two systems in Ag trapping, signal reading, and standard calibration.

In summary, through large-scale microarray analyses, we analyzed the gene expression profiles of human pulmonary epithelial cells exposed to varied doses of alcohol. GILZ, a key GC-responsive gene, responds to alcohol in a dose-dependent manner. These data suggest, for the first time, that alcohol and GCs share the same signaling pathway to modulate cellular inflammatory and immune propensities.

Disclosures The authors have no financial conflicts of interest.