Background: Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGFβ1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGFβ1 and GM-CSF and thereby alter airway epithelial barrier function. Methods: Human or rat LF were cultured ± ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFβ1 receptor type 1 (TGFβR1) inhibitor (SB431542) or a TGFβ1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGFβ1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA. Results: AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and α-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGFβ1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFβ1 activity. Conclusions: Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFβ1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.

Background: Alcohol impairs pulmonary innate immune function and is associated with an increased risk for tuberculosis (TB). Toll-like receptor 2 (TLR2) is a pattern recognition receptor on alveolar macrophages that recognizes Mycobacterium tuberculosis (Mtb). Its expression depends, in part, on granulocyte macrophage-colony stimulating factor (GM-CSF) signaling. Given our prior work demonstrating suppression of GM-CSF signaling in chronic alcohol ingestion, we hypothesized that alcohol impairs TLR2 expression via suppression of GM-CSF and thereby reduces the ability of the macrophage to recognize and phagocytose Mtb. Methods: Primary alveolar macrophages were isolated from control-fed and alcohol-fed rats. Prior to cell isolation, some alcohol-fed rats were treated with intranasal GM-CSF and then endotracheally inoculated with an attenuated strain of Mtb. Primary macrophages were then isolated and immunofluorescence (IF) was used to determine phagocytic efficiency and TLR2 expression in the presence and absence of GM-CSF treatment as well as phagocytic efficiency in the presence and absence of TLR2 neutralization. Results: TLR2 expression and phagocytosis of Mtb were significantly decreased in the alveolar macrophages of alcohol-fed rats compared to control-fed rats. In parallel, blocking TLR2 signaling recapitulated this decreased phagocytosis of Mtb. In contrast, intranasal GM-CSF treatment restored TLR2 expression and Mtb phagocytosis in the alveolar macrophages of alcohol-fed rats to levels comparable to control-fed rats. Conclusions: Chronic alcohol ingestion reduces TLR2 protein expression and phagocytosis of Mtb, likely as a result of impaired GM-CSF signaling. GM-CSF restores membrane-bound TLR2 expression and phagocytic function.

Macrophage phenotype and function is dependent on the underlying microenvironment. Many diseases are accompanied by abnormal shifts in macrophage polarization state that limit the ability of the cells to become innate immune effectors. Previous work in the field suggests that chronic alcohol ingestion, which is associated with a shift away from innate immune effector macrophages, is also associated with a deficient response to oxidative stress. We therefore hypothesized that the optimal response to oxidative stress was dependent on the ability of the macrophage to become an innate immune effector cell. To investigate this hypothesis, we first confirmed that we could reproducibly polarize NR8383 cells (a rat alveolar macrophage cell line) into the prototypical M1 and M2 states (using IFN-γ and IL-4, respectively). We then tested the polarized cells for their ability to scavenge reactive oxygen species generated by glucose oxidase (GOX) using the Amplex red assay and found that IFN-γ-polarized cells had greater scavenging capacity. To elucidate the mechanism of the enhanced response to oxidative stress, we then assessed key components of the anti-oxidant response; specifically, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the master transcription factor responsible for the cellular response to oxidative stress, and one of its downstream effectors, glutamate-cysteine ligase catalytic subunit (GCLC). We found that both proteins were significantly upregulated in the IFN-γ-polarized cells. To confirm that Nrf2 is an integral component of this improved anti-oxidant response, we transfected IFN-γ-polarized cells with either silencing RNA to Nrf2 or control silencing RNA and found that hydrogen peroxide scavenging was significantly impaired in the si-Nrf2-treated cells. Further, transfecting untreated cells with si-Nrf2 polarized them toward the M2 phenotype in the absence of IL-4, suggesting a mechanistic role for Nrf2 in macrophage polarization. We then confirmed several of our key experiments in primary rat alveolar macrophages cells. Taken together, these findings suggest that the M1 polarization state is necessary for the optimal response to oxidative stress in the macrophage, and that this response is mediated through Nrf2 and its downstream effectors.

Background: Alcohol significantly impairs antioxidant defenses and innate immune function in the lung and increases matrix metalloproteinase 9 (MMP-9) activity. The receptor for advanced glycation end products (RAGE) is a well-characterized marker of lung injury that is cleaved by MMP-9 into soluble RAGE and has not yet been examined in the alcoholic lung. We hypothesized that chronic alcohol ingestion would impair RAGE signaling via MMP-9 in the alveolar macrophage and thereby impair innate immune function. Materials and Methods: Primary alveolar macrophages were isolated from control-fed or alcohol-fed rats. Real-time polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assays were performed to evaluate RAGE expression. Silencing of MMP-9 ribonucleic acid (RNA) in a rat alveolar macrophage cell line was confirmed by qRT-PCR, and immunofluorescence (IF) was used to assess the association between alcohol, MMP-9, and RAGE. Phagocytosis was assessed using flow cytometry. Sulforaphane and glutathione were used to assess the relationship between oxidative stress and RAGE. Results: RAGE messenger RNA expression was significantly increased in the alveolar macrophages of alcohol-fed rats, but IF showed that membrane-bound RAGE protein expression was decreased. Lavage fluid demonstrated increased levels of soluble RAGE (sRAGE). Decreasing MMP-9 expression using si-MMP-9 abrogated the effects of alcohol on RAGE protein. Phagocytic function was suppressed by direct RAGE inhibition, and the impairment was reversed by antioxidant treatment. Conclusions: Chronic alcohol ingestion reduces RAGE protein expression and increases the amount of sRAGE in alveolar lavage fluid, likely via cleavage by MMP-9. In addition, it impairs phagocytic function. Antioxidants restore membrane-bound RAGE and phagocytic function.

Background: Alcohol abuse increases the risk for acute lung injury (ALI). In both experimental models and in clinical studies, chronic alcohol ingestion causes airway oxidative stress and glutathione depletion and increases the expression of transforming growth factor beta-1 (TGFβ1), a potent inducer of fibrosis, in the lung. Therefore, we hypothesized that alcohol ingestion could promote aberrant fibrosis following experimental ALI and that treatment with the glutathione precursor s-adenosylmethionine (SAMe) could mitigate these effects. Methods: Three-month-old C57BL/6 mice were fed standard chow ± alcohol (20% v/v) in their drinking water for 8 weeks and ±SAMe (4% w/v) during the last 4 weeks. ALI was induced by intratracheal instillation of bleomycin (2.5 units/kg), and lungs were assessed histologically at 7 and 14 days for fibrosis and at 14 days for the expression of extracellular matrix proteins and TGFβ1. Results: Alcohol ingestion had no apparent effect on lung inflammation at 7 days, but at 14 days after bleomycin treatment, it increased lung tissue collagen deposition, hydroxyproline content, and the release of activated TGFβ1 into the airway. In contrast, SAMe supplementation completely mitigated alcohol-induced priming of these aberrant fibrotic changes through decreased TGFβ1 expression in the lung. In parallel, SAMe decreased alcohol-induced TGFβ1 and Smad3 mRNA expressions by lung fibroblasts in vitro. Conclusions: These new experimental findings demonstrate that chronic alcohol ingestion renders the experimental mouse lung susceptible to fibrosis following bleomycin-induced ALI, and that these effects are likely driven by alcohol-mediated oxidative stress and its induction and activation of TGFβ1. © 2013 by the Research Society on lcoholism.

Both mechanical loading and nitric oxide (NO) have positive influences on bone mass. NO production is induced by mechanical strain via upregulation of eNOS mRNA and protein, the predominant NOS in adult bone. At the same time, strain causes decreased expression of RANKL, a factor critical for osteoclastogenesis. In this study, we harvested primary stromal cells from wild-type (WT) and eNOS(−/−) mice to test whether induction of NO by mechanical strain was necessary for transducing mechanical inhibition of RANKL. We found that strain inhibition of RANKL expression was prevented by NOS inhibitors (L-NAME and L-NMMA) in WT stromal cells. Surprisingly, stromal cells from eNOS(−/−) mice showed significant mechanical repression of RANKL expression (p<0.05). Mechanical strain still increased NO production in the absence of eNOS, and was abolished by SMTC, a specific nNOS inhibitor. nNOS mRNA and protein expression were increased by strain in eNOS(−/−) but not in WT cells, revealing that nNOS was mechanically sensitive. When NO synthesis was blocked with either SMTC or siRNA targeting nNOS in eNOS(−/−) cells however, strain still was able to suppress RANKL expression by 34%. This indicated that strain suppression of RANKL can also occur through non-NO dependent pathways. While our results confirm the importance of NO in the mechanical control of skeletal remodeling, they also suggest alternative signaling pathways by which mechanical force can produce anti-catabolic effects on the skeleton.

Background:HIV is associated with an increased risk for emphysema. Matrix metalloproteinase 9 (MMP-9) is a lung tissue remodeling enzyme associated with emphysema. We previously found MMP-9 activity increases with increases in oxidative stress and that HIV increases alveolar oxidative stress. We hypothesized that HIV proteins would increase the risk of cigarette smoke-induced emphysema due to MMP-9.Methods:HIV-1 transgenic rats and wild-type littermates were exposed to cigarette smoke or sham for 8 weeks. Lung compliance and histology were assessed. Bronchoalveolar lavage (BAL), primary alveolar macrophages (AM), and serum samples were obtained. A rat alveolar macrophage cell line was exposed to the HIV protein Tat, and MMP-9 levels were assessed by Western immunoblotting. MMP-9 protein expression and activity were assessed in AM from the HIV rat model by ELISA and cytoimmunofluoresence, respectively. Serum from human subjects with and without HIV and tobacco dependence was assessed for MMP-9 levels.Results:MMP-9 expression was significantly increased in rat alveolar macrophages after Tat exposure. HIV-1 transgenic rats developed emphysema while wild-type littermates did not. MMP-9 expression was also increased in the serum, BAL, and AM of HIV-1 transgenic rats after exposure to cigarette smoke compared with wild-type rats. In parallel, serum samples from HIV+ smokers had higher levels of MMP-9 than subjects without HIV and those who did not smoke.Conclusion:The combination of HIV and cigarette smoke increases MMP-9 expression in experimental rat HIV models and human subjects. HIV and cigarette smoke both induce alveolar oxidative stress and thereby increase MMP-9 activity.

Background Alcohol abuse and HIV-1 infection frequently co-exist and these individuals are at high risk for serious lung infections and respiratory failure. Although alcohol ingestion and HIV-1 transgene expression have been shown to independently cause oxidative stress and disrupt alveolar epithelial barrier function in experimental models, their interactive effects have not been examined. Methods and Results In this study we determined that chronic alcohol ingestion (12 wks) exacerbated the already significant defects in alveolar epithelial paracellular permeability and lung liquid clearance in HIV-1 transgenic rats. Further, immunocytochemical analyses of tight junction protein expression in primary alveolar epithelial cells showed that occludin and zonula occludens-1 (ZO-1) localization within the plasma membrane was more disrupted than in either condition alone, consistent with the observed defects in epithelial barrier function. Interestingly, expression of Nrf2, the transcription factor required to activate the antioxidant response element, was decreased in primary alveolar epithelial cells isolated from HIV-1 transgenic rats. In parallel, exposing lung epithelial cells in vitro to either alcohol or the HIV-related protein gp120 also decreased Nrf2 expression. Importantly, treatment with procysteine, which increases thiol antioxidants including glutathione, improved tight junction protein localization in the plasma membrane and restored alveolar epithelial barrier function in alcohol-fed HIV-1 transgenic rats. Conclusions These results provide novel evidence that HIV-related proteins and alcohol together causes more barrier dysfunction in the lung epithelium than either stress alone. However, these significant effects on the alveolar barrier can be mitigated by augmenting the thiol antioxidant pool, a strategy with potential clinical applications in subjects who are highly vulnerable to lung disease because of co-existent alcohol abuse and HIV infection.

Background Chronic alcohol abuse causes oxidative stress, impairs alveolar macrophage immune function, and increases the risk of pneumonia and acute lung injury. Recently we determined that chronic alcohol ingestion in rats decreases zinc levels and macrophage function in the alveolar space; provocative findings in that zinc is essential for normal immune and antioxidant defenses. Alveolar macrophage immune function depends on stimulation by GM-CSF, which signals via the transcription factor PU.1. In parallel, the antioxidant response element signals via the transcription factor Nrf2. However, the role of zinc bioavailability on these signaling pathways within the alveolar space is unknown. Methods To determine the efficacy of dietary zinc supplementation on lung bacterial clearance and oxidative stress, we tested three different groups of rats: control-fed, alcohol-fed, and alcohol-fed with zinc supplementation. Rats were then inoculated with intratracheal Klebsiella pneumoniae and lung bacterial clearance was determined 24 hrs later. Isolated alveolar macrophages were isolated from uninfected animals and evaluated for oxidative stress and signaling through PU.1 and Nrf2. Results Alcohol-fed rats had a 5-fold decrease in lung bacterial clearance compared to control-fed rats. Dietary zinc supplementation of alcohol-fed rats normalized bacterial clearance and mitigated oxidative stress in the alveolar space, as reflected by the relative balance of the thiol redox pair cysteine and cystine, and increased nuclear binding of both PU.1 and Nrf2 in alveolar macrophages from alcohol-fed rats. Conclusions Dietary zinc supplementation prevents alcohol-induced alveolar macrophage immune dysfunction and oxidative stress in a relevant experimental model, suggesting that such a strategy could decrease the risk of pneumonia and lung injury in individuals with alcohol use disorders.

Previously we determined that chronic alcohol ingestion (6 wks) in rats increases lung epithelial permeability in vivo ~5-6-fold and promotes flooding of the alveolar airspaces with proteinaceous fluid in response to stresses such as sepsis. In parallel, alveolar epithelial cells isolated from alcohol-fed rats fail to form tight monolayers in vitro, even when cultured for up to 8 days in the absence of alcohol. However, the molecular mechanisms underlying alcohol-induced permeability are unknown. Claudins are key components of tight junctions that restrict the paracellular movement of water, proteins, and solutes across cellular barriers including the alveolar epithelium. In this study, we examined the expression of multiple members of the claudin protein family in the lungs of alcohol-fed vs. control-fed rats (Lieber-DeCarli liquid diet with 36% of calories as alcohol vs. maltin-dextrin for 6 wks). We determined that chronic alcohol ingestion affected the expression of multiple claudins; most striking were decreases in claudin-1 and claudin-7, and an increase in claudin-5, in the whole lung and in alveolar epithelial monolayers derived from alcohol-fed rats. In parallel, immunocytochemistry of alveolar epithelial monolayers from alcohol-fed rats revealed abnormal intracellular accumulation of claudin-7 protein and relatively decreased localization to cell membranes. Claudin-1 and claudin-7 are relatively specific to alveolar epithelial type I pneumocytes that form the vast majority of the alveolar epithelial barrier in vivo, and increases in claudin-5 have been associated with increased epithelial permeability in other systems. Therefore, these findings suggest that changes in claudin expression in the alveolar epithelium produce a “leakier” phenotype that renders the alcoholic lung susceptible to alveolar flooding during acute inflammatory stresses.