Alcohol exposure alters mouse lung inflammation in response to inhaled dust

Abstract

Alcohol exposure is associated with increased lung infections and decreased mucociliary clearance. Occupational workers exposed to dusts from concentrated animal feeding operations (CAFOs) are at risk for developing chronic inflammatory lung diseases. Agricultural worker co-exposure to alcohol and organic dust has been established, although little research has been conducted on the combination effects of alcohol and organic dusts on the lung. Previously, we have shown in a mouse model that exposure to hog dust extract (HDE) collected from a CAFO results in the activation of protein kinase C (PKC), elevated lavage fluid cytokines/chemokines including interleukin-6 (IL-6), and the development of significant lung pathology. Because alcohol blocks airway epithelial cell release of IL-6 in vitro, we hypothesized that alcohol exposure would alter mouse lung inflammatory responses to HDE. To test this hypothesis, C57BL/6 mice were fed 20% alcohol or water ad libitum for 6 weeks and treated with 12.5% HDE by intranasal inhalation method daily during the final three weeks. Bronchoalveolar lavage fluid (BALF), tracheas and lungs were collected. HDE stimulated a 2-4 fold increase in lung and tracheal PKCε (epsilon) activity in mice, but no such increase in PKCε activity was observed in dust-exposed mice fed alcohol. Similarly, alcohol-fed mice demonstrated significantly less IL-6 in lung lavage in response to dust than that observed in control mice instilled with HDE. TNFα levels were also inhibited in the alcohol and HDE-exposed mouse lung tissue as compared to the HDE only exposed group. HDE-induced lung inflammatory aggregates clearly present in the tissue from HDE only exposed animals were not visually detectable in the HDE/alcohol co-exposure group. Statistically significant weight reductions and 20% mortality were also observed in the mice co-exposed to HDE and alcohol. These data suggest that alcohol exposure depresses the ability of the lung to activate PKCε-dependent inflammatory pathways to environmental dust exposure. These data also define alcohol as an important co-exposure agent to consider in the study of inhalation injury responses.

Keywords: alcohol; inflammation; mortality; organic dust.

Figure 1

C57BL/6 Mice were fed alcohol (EtOH) for 6 weeks and were exposed to 12.5% hog barn dust extract (HDE) intranasally for 3 weeks (n = 5). Body weights of the alcohol + HDE group were statistically lower as compared to control group weights (17.9/20.1 g) (* p < 0.05 vs. control ANOVA, Bonferroni post-test).

Figure 2

C57BL/6 Mice were fed alcohol (EtOH) for 6 weeks and were exposed to 12.5% HDE intranasally for 3 weeks (n = 5). (A) Total cells counted in BALF: Total cellularity collected in the bronchoalveolar lavage fluid (BALF) increased nearly 10-fold in the HDE and alcohol + HDE groups however there is no statistical difference between the HDE and co-exposure group. (B) Total cells counted in BALF per cell type: Inflammatory cells quantified from BALF detailed a statistical increase in neutrophils in the HDE group, which is followed by a reduction in neutrophils in the alcohol + HDE group. Inflammatory cells quantified from BALF detailed a difference in distribution between alcohol + HDE and the HDE group alone. (C) Percentage of each type of cells coutnted: Neutrophils increased from less than 1% in the control to 65% in the HDE group and back down to 37% in the alcohol + HDE group. (* p < 0.05 vs. control ANOVA, Bonferroni post-test) (M = macrophages, P = neutrophils, EO = eosinophils, L = lymphocytes).

Figure 3

C57BL/6 Mice were fed alcohol (EtOH) for 6 weeks and were exposed to 12.5% HDE intranasally for 3 weeks (n = 5). BAL fluid was collected from these mice and cytokine levels were quantified via ELISA. (A) IL-6 was ~50% less in the alcohol + HDE group as opposed to the HDE only group (46.8/96.1 pg/mL);(B) KC quantification detailed a statistically significant reduction in the alcohol + HDE group compared to HDE only group (34.1/79.6 pg/mL); (C) MIP-2 quantification detailed a statistically significant reduction in the alcohol + HDE vs. the HDE only group (23.1/31.5 pg/mL) (* p < 0.05 vs. control ANOVA, Bonferroni post-test; ^#p < 0.05 vs. HDE, ANOVA, Bonferroni post-test).

Figure 4

C57BL/6 Mice were fed alcohol (EtOH) for 6 weeks and were exposed to 12.5% HDE intranasally for 3 weeks (n = 5). Cytokine in the BAL fluid collected from these mice was quantified by ELISA. HDE instillation elevated TNF release more than 3-fold vs. control (* p < 0.05 vs. control, ANOVA, Bonferroni post-test). TNF-α levels were reduced by 65% in the alcohol + HDE group compared to the HDE only group (117.6/334.1 pg/mL). (^#p < 0.05 vs. HDE, ANOVA, Bonferroni post-test).

Figure 5

C57BL/6 Mice were fed alcohol (EtOH) for 6 weeks and were exposed to 12.5% HDE intranasally for 3 weeks (n = 5). Protein kinase activity was quantified from the tracheal epithelium removed from these mice. (A) PKCε activity in the tracheal epithelial cells of the alcohol + HDE group was statistically significantly lower as compared to the HDE only group (622.2/1255.5 pmol/mg/min) (^#p < 0.05); (B) PKCα activity in tracheal epithelial cells was unchanged in the epithelial cells unchanged in the treatment groups; (C) PKA activity in the tracheal epithelial cells of the alcohol + HDE treated mice was ~50% lower than the HDE only treated mice (261.1/544.6 pmol/mg/min) (^#p < 0.05).

Figure 6

C57BL/6 Mice were fed alcohol (EtOH) for 6 weeks and were exposed to 12.5% HDE intranasally for 3 weeks (n=5). Protein kinase activity was quantified from the ex-vivo lung tissue removed from C57Bl/6 mice. (A) PKCε activity in the lung slices of the alcohol+HDE treated tissue was ~80% less than the activity of observed in the HDE only group (404.1/2058.5 pmol/mg/min);(B) PKCα activity in the lung slices of C57Bl/6 mice treated with alcohol+HDE and HDE only were both statistically higher than the control;(C) PKA activity was unchanged between treatment groups (* ^#p< 0.05 vs. control ANOVA, Bonferroni post-test).

Figure 7

C57BL/6 Mice were fed alcohol (EtOH) for 6 weeks and were exposed to 12.5% HDE intranasally for 3 weeks (n = 5). Lung sections from H&E stained slides revealed upon histological evaluation that HDE exposed C57BL/6 mice exhibited the highest levels of peribronchial inflammation. The alcohol + HDE exposed group showed a marked reduction in HDE-induced mononuclear cellular aggregates.

Figure 8

Proposed mechanism by which alcohol exposure ablates HDE-induced inflammation.