All animal procedures were conducted in accordance with the rules and regulations of the Institutional Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) under the protocol LPS-GK1. Twelve-week-old male C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME, United States). Mice were exposed to either control or ethanol-containing Lieber-DeCarli liquid diet. The NIAAA model of chronic and binge ethanol feeding was followed as described (Bertola et al., 2013). In brief, mice were fed a control liquid diet for 5 days, after which they divided into two groups: a pair-fed control group and an ethanol-fed group. For the next 10 days, the ethanol-fed group received a liquid diet containing 5% ethanol, whereas the pair-fed control group received an isocaloric control liquid diet. On day 11, the pair-fed control mice received maltose dextrin via gavage, and the ethanol-fed mice received 5 g/kg ethanol by gavage. The experimental paradigm is depicted in Figure 1A. Four- five mice used per group.

Following the gavage, mice were sacrificed by decapitation. Serum was obtained from by retroorbital bleeding. Left lung tissue was snap-frozen in liquid nitrogen for protein and mass spectrometry analysis. Portions of right lung tissue were placed in RNAlater (ThermoFisher Scientific) and were kept at 4°C overnight for RNA extraction.

Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were measured using EnzyChrom™ Alanine Transaminase Assay Kit and EnzyChrom™ Aspartate Transaminase Assay Kit, respectively (BioAssay Systems).

RNA extraction was performed using RNeasy Mini Kits from Qiagen (Valencia, CA). One microgram of total RNA was reverse transcribed to cDNA using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Expression of the target genes was quantified using gene-specific primers and PowerSYBRGreen master mix (ThermoFisher Scientific) in a QuantStudio 3 Real-Time PCR instrument (ThermoFisher Scientific). Predesigned mouse Alox15 (QT00111034), Ptgs1 (QT00155330), Ptgs2 (QT00165347), Nox1 (QT00140091), Nox4 (QT00126042), Aldh2 (QT00158368), Adh1 (QT00093520), Cyp2e1 (QT00112539), Cybb (QT00139797) primers were purchased from Qiagen (Valencia, CA). Fold changes in gene expression relative to the house-keeping gene TATA-Box Binding Protein (Tbp, QT00198443) were determined.

Levels of phosphatidylcholines (DPPC, PAPC, POPC) and truncated oxidized phosphatidylcholines (PGPC, POVPC, PAzePC, 16-9-ALDOPC) were measured in lungs by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Left lung tissue was homogenized in 0.5 ml of ice-cold methanol/Tris buffer (50 mM, pH 8.0), 1:1, containing 250 ng of [²H₄] Platelet Activating Factor C-16 ([²H₄] PAF C-16) as internal standard. Homogenates were extracted three times with CHCl₃: MeOH (2:1, vol/vol), dried under nitrogen flow and reconstituted with MeOH after precipitating proteins with ice-cold acetone. LC-MS/MS analyses were conducted on an Agilent 6,470 triple quadrupole mass spectrometer (Agilent Technologies) coupled to an Agilent 1200 LC system. Analytes were separated using an Agilent InfinityLab Poroshell 120 EC-C18 column (3.0 × 100 mm, 2.7 micron). Gradient elution mobile phases consisted of 0.1% formic acid in H₂O (phase A) and 0.1% formic acid in MeOH (phase B). Gradient elution (350 μL/min) was initiated at 10% B followed by a linear increase to 50% B at 0.5 min and then another linear increase to 85% B at 3 min and maintained until 14 min before increasing linearly to 100% B by 18 min and maintained until 38 min. The mass spectrometer was set for Agilent Jet Stream high sensitivity electrospray ionization operated in positive ion mode. The source parameters were as follows: capillary voltage, 3500 V; gas temperature, 300°C; drying gas flow, 5 L/min; sheath gas temperature, 250°C; sheath gas flow, 10 L/min. Nitrogen was used as the nebulizing gas. Collision-induced dissociation was performed using nitrogen. Levels of each compound were analyzed by multiple reaction monitoring. The molecular ion and fragment for each compound were measured as follows: m/z 734.6→183.9 for DPPC, m/z 782.6→183.9 for PAPC, m/z 746.6→183.9 for POPC, m/z 610.4→183.9 for PGPC, m/z 594.4→183.9 for POVPC, m/z 650.4→183.9 for 16-9-ALDOPC, m/z: 482.4 →104.1 for Lyso-PAF C16:0, m/z:524.4 →183.9 for PAF C16:0 m/z 666.4→183.9 for PAzePC, m/z 528.4→184 for [²H₄]PAF C16:0. Analytes were quantified using MassHunter Workstation LC/QQQ Acquisition and MassHunter Workstation Quantitative Analysis software (Agilent Technologies). Levels of lipids in the samples were measured against standard curves.

To induce acute lung injury, 16-week-old mice received a single oropharyngeal instillation of lipopolysaccharide (LPS) (50 µg/mouse) as described previously (Park et al., 2020). LPS (L4391, Millipore Sigma) was dissolved in sterile saline and administered at 50 µg/mouse in 40 µL after anesthesia induced by ketamine/xylazine. In this part of the study both pair-fed control and ethanol-fed mice received sterile saline. The mice were studied 24 h after LPS or saline instillation, as depicted schematically in Figure 4A.

BALF was collected from anesthetized mice by lavaging lungs three times with 0.8 ml Hanks’ balanced salt solution without calcium and magnesium (HBSS) (Sigma Aldrich). Supernatants were collected as BALF for further analyses.

CyQuant LDH cytotoxicity assay kit (Invitrogen) was used to assess LDH activity in BALF samples, as detailed in the product manual.

Albumin levels were measured by Mouse Albumin Elisa Assay kit obtained from Bethyl Laboratories. BALF samples from pair-fed control and ethanol-fed mice were diluted at 1:1000 ratio, whereas samples from LPS group were diluted at 1:2000 ratio.

Total protein in BALF samples was measured using Pierce BCA protein assay kit as detailed in the product manual (ThermoFisher Scientific).

Levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β) and Interleukin 17 (IL17) in mouse BALF were measured using the mouse CCL2 quantikine ELISA Kit, (R&D systems, MJE00B), mouse TNF-alpha quantikine ELISA Kit, (R&D systems, MTA00B), mouse IL-1β quantikine ELISA Kit, (R&D systems, MLB00C), mouse IL-17 quantikine ELISA Kit, (R&D systems, M1700), respectively.

Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software Inc.). Two-way ANOVA followed by Sidak’s multiple comparisons test was performed. p < 0.05 was considered significant.

The NIAAA mouse model of chronic with binge ethanol feeding was utilized in this study because the model is known to better induce liver injury, inflammation and fatty liver, as compared to ethanol feeding models without addition of a binge or secondary insult. The chronic with binge model is also thought to better mimic the drinking patterns of people with an AUD (Bertola et al., 2013). To verify that the ethanol feeding model induced liver injury, we measured serum alanine transaminase (ALT) and aspartate transaminase (AST) levels in pair-fed (PF) control mice and ethanol-fed (EtOH) mice. ALT and AST were significantly elevated in the serum of EtOH mice as compared to PF mice at the 9-h time point only (Figure 1B), indicating significant acute liver injury, which is then mostly resolved by 24-h post-Ethanol binge.

Increased oxidative stress has been postulated as a possible mechanism of alcohol-induced lung injury (Guidot and Roman, 2002a; Massey et al., 2015a). We therefore measured gene expression of oxidative enzymes known to be involved in oxidative stress, such as the inflammatory oxidative enzymes arachidonate 15-lipoxygenase (Alox15), prostaglandin synthase 1 and 2 (Ptgs1 and Ptgs2), and NADPH oxidase 1, 2 and 4 (Nox1, Nox2 and Nox4). Alox15 gene expression was significantly increased 9 h after the binge and then remained elevated for 24 h (Figure 2A). Ptgs2 gene expression was only increased at 24 h after the binge whereas no change was observed in Ptgs1 expression (Figure 2B). Nox1 expression was significantly increased and Nox2 was decreased at 9 h after the binge, which then normalized at 24 h post-binge, whereas we have not observed any changes in Nox4 expression in lung post-binge (Figure 2C). Multiple alcohol metabolizing enzymes such as alcohol dehydrogenase 1 (Adh1), alcohol dehydrogenase 2 (Aldh2) and Cytochrome P450 2E1 (Cyp2E1) are functionally expressed in lungs (Oyama et al., 2005; Mouton et al., 2016). They may also contribute oxidative stress in lungs in addition to alcohol metabolism (Kaphalia et al., 2014). We found that alcohol has not changed gene expression levels of Adh1 and Aldh2 (Figure 2D) whereas expression level of Cyp2e1 is significantly increased (Figure 2E). These results indicate increased oxidative stress in lungs following chronic ethanol and single binge. Since we found significant and sustained elevation in gene expression of several oxidative enzymes, we further explored its functional consequences on lung phospholipid homeostasis.

Surfactant phospholipid homeostasis is critical for lung function and host defense. Surfactant phospholipids predominantly consist of dipalmitoyl phosphatidylcholine (DPPC) that about 60% of surfactant lipids in the lungs consist of DPPC. Besides, cellular phospholipids with mono or poly unsaturated fatty acid moieties such as palmitoyl arachidonoyl phosphatidylcholine (PAPC), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl linoleoyl phosphatidylcholine (PLPC) could also involve multiple inflammatory and oxidative processes. Previously, it was shown that chronic alcohol feeding for 6–8 weeks increased levels of phosphatidylcholines (Liau et al., 1981). Using the NIAAA model, we found that DPPC was significantly increased 9 h after the single binge (Figure 3A). Although there is a slight trend towards increased levels of PAPC and POPC, these changes are not statistically significant (Figure 3A).

One of the consequences of an ethanol-induced increase in oxidative stress and in the expression of Alox15 in lungs would be lipid oxidation and generation of truncated oxidized phospholipids (tOxPLs). tOxPLs are bioactive and can have harmful effects on tissues (Karki and Birukov, 2020). For example, the tOxPLs of PAPC (lyso-PC, POVPC, and PGPC) increased endothelial cell permeability in a dose-dependent manner and were barrier disruptive (Birukova et al., 2013). Importantly, tOxPLs could contribute to lung injury, and they are lipid mediators of acute respiratory distress syndrome in lungs (Ke et al., 2019). To explore the status of tOxPLs in lungs in the NIAAA model, we measured the levels of tOxPLs such as palmitoyl oxovaleryl phosphatidyl choline (POVPC), palmitoyl glutaryl phosphatidyl choline (PGPC), palmitoyl oxo-nonanoyl phosphatidyl choline (ALDO-PC), and palmitoyl azelaoyl phosphatidyl choline (PAzePC). POVPC and PGPC are the tOxPLs of surfactant phospholipid PAPC, while ALDO-PC and PAzePC are the tOxPLs of surfactant phospholipid POPC and PLPC. At the 9-h time point, ethanol significantly increased the levels of POVPC, PGPC, ALDO-PC, and PAzePC in the lungs as compared to control lungs (Figure 3B). At the 24-h time point, mild but not significant increases in the levels of all four tOxPLs were observed (Figure 3B). Truncated oxidized phospholipids are structural analogue of Platelet activator factors (PAFs) and Lyso-PAFs. Indeed, there are evidence that they bind to PAF receptors. Therefore, we also check the levels of PAFs. Alcohol ingestion has not changed the levels of PAF C16:0 (Figure 3C) or lyso-PAF C16:0 (Figure 3D). These results suggest that oxidative stress transiently increases tOxPLs, which act as inflammatory lipid mediators in the lungs of ethanol-fed mice.

Hallmarks of acute lung injury include increased vascular permeability, cytotoxicity, and disrupted epithelial and cell membrane integrity (Matute-Bello et al., 2008). It is known that heavy alcohol drinking in individuals with AUD increases vascular permeability and dysregulates epithelial integrity in lungs (Joshi and Guidot, 2007; Burnham et al., 2009). To explore the status of acute lung injury in the NIAAA model, we collected bronchoalveolar lavage fluid (BALF) from mice 9 h after the ethanol binge feeding and measured albumin, lactate dehydrogenase (LDH) and total protein in BALF as indicators of increased vascular permeability, cytotoxicity and disrupted epithelial integrity. For comparison, we made similar measurements in the BALF of mice following oropharyngeal LPS instillation, an established model of acute lung injury. Levels of albumin, total protein and LDH were not altered in BALF by 10 days chronic ethanol followed by a single binge (Figure 4), whereas LPS significantly increased albumin, total protein and LDH activity in BALF (Figures 4A–C), indicating acute lung injury.

To explore status of inflammatory cytokines in lung after either chronic feeding plus single ethanol binge or LPS instillation, we measured inflammatory cytokines in BALF. LPS instillation significantly increased the BALF content of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β) and Interleukin 17 (IL17) (Figures 4B–D). in contrast, ethanol did not significantly alter the BALF levels of MCP-1, TNFα and IL-1β (Figures 4E–G) but significantly increased IL17 to levels seen after LPS instillation (Figure 4H).