Alcohol primes the airway for increased interleukin-13 signaling

Abstract

Background: Using an experimental model of airway fibrosis following lung transplantation, we recently showed that chronic alcohol ingestion by donor rats amplifies airway fibrosis in the recipient. Associated with alcohol-mediated amplification of airway fibrosis is increased transforming growth factor beta-1(TGFbeta(1)) and alpha-smooth muscle actin expression. Other studies have shown that interleukin-13 (IL-13) modulates TGFbeta(1) signaling during experimentally-induced airway fibrosis. Therefore, we hypothesized that IL-13 is a component of alcohol-mediated amplification of pro-fibrotic mediators in the alcoholic lung.

Methods: To test this hypothesis, we analyzed tracheal epithelial cells and type II alveolar cells from control- or alcohol-fed rats, alcohol-treated mouse lung fibroblasts, and human bronchial epithelial cells in vitro for expression of various components of the IL-13 signaling pathway. Signaling via the IL-13 pathway was assessed by measuring levels of phosphorylated signal transducers and activators of transcription-6 (STAT6). In addition, we performed heterotopic tracheal transplantation using control-fed and alcohol-fed donor rats and analyzed tracheal allografts for expression of components of the IL-13 signaling pathway by RT-PCR and immunocytochemical analyses.

Results: Interleukin-13 expression was detected in type II alveolar epithelial cells and human bronchial epithelial cells, but not in lung fibroblasts. IL-13 expression was decreased in whole lung and type II cells in response to alcohol exposure. In all cell types analyzed, expression of IL-13 signaling receptor (IL-13R alpha(1)) mRNA was markedly increased. In contrast, mRNA and protein expression of the IL-13 decoy receptor (IL-13R alpha(2)) were decreased in all cells analyzed. Exposure to alcohol also increased STAT6 phosphorylation in response to IL-13 and lipopolysaccharide.

Conclusions: Data from multiple cell types in the pulmonary system suggest that IL-13 and its receptors play a role in alcohol-mediated activation of pro-fibrotic pathways. Taken together, these data suggest that alcohol primes the airway for increased IL-13 signaling and subsequent tissue remodeling upon injury such as transplantation.

Figure 1. Chronic alcohol exposure in rats increases expression of IL-13Rα₁ and decreases IL-13Rα₂ expression in the whole lung

(A) Immuno-staining for IL-13Rα₂ in whole lung sections from control- (panel i) or alcohol-fed (panel ii) rats. Chronic alcohol exposure decreases IL-13Rα₂ protein expression in cells lining the alveolar walls (panels i and ii). (B) RT-PCR analyses of mRNA expression in whole lung for components of IL-13-mediated signaling. (C) Quantitation of RT-PCR results. IL-13Rα₁ expression is increased 2.5-fold whereas IL-13 and IL-13Rα₂ expression is decreased 49% and 46%, respectively in lungs from alcohol-fed rats. 18S rRNA was used as a control for RNA input. Immuno-staining and RT-PCR images are representative of lungs from 3 animals for each group. Bar = 100 μm. * p < 0.05 compared to Ctrl for each gene.

Figure 2. Chronic alcohol exposure in rats increases components of pro- IL-13 signaling in type II alveolar epithelial (AT2) cells and primary lung fibroblasts

(A) Immuno-staining for IL-13Rα₂ in AT2 cells from control- (panel i) or alcohol-fed (panel ii) rats. Chronic alcohol exposure decreases IL-13Rα₂ protein expression. (B) RT-PCR analyses of mRNA expression in AT2 cells for components of IL-13-mediated signaling. (C) Quantitation of RT-PCR analyses in AT2 cells. AT2 cells express IL-13 and expression is decreased 40% in cells from alcohol-fed rats. IL-13Rα₁ expression is increased 2.5-fold and IL-13Rα₂ expression is decreased 49% compared to AT2 cells from control-fed rats. (D) Expression of components of IL-13-mediated signaling in primary lung fibroblasts after 48 hr incubation in the presence of 60 mM or 50 μg/ml nicotine. (E) Quantitation of RT-PCR analyses in primary lung fibroblasts. IL-13Rα₁ expression is increased 4.3-fold and IL-13 Rα₂ expression in decreased 57% in response to alcohol. 18S rRNA was used as a control for RNA input. RT-PCR images are representative of 3 independent experiments. Immuno-staining and RT-PCR images are representative of 3 independent experiments. Bar = 50 μm. *^, ** p < 0.05 compared to Ctrl for each gene.

Figure 3. Alcohol exposure increases expression of pro-IL-13 signaling components in epithelial cells of the conducting airways

(A) Immuno-staining for IL-13Rα₂ in tracheal sections from control- (Ctrl) or alcohol- (EtOH) fed rats. Chronic alcohol exposure decreases expression of IL-13Rα₂ protein in the tracheal epithelium. (B) RT-PCR analyses of mRNA expression in cultured Beas2B cells for components of IL-13-mediated signaling. (C) Quantitation of RT-PCR results in Beas2B cells. Exposure to 60 mM alcohol for 48 hr increases IL-13 expression by 43%. In contrast, expression of IL-13Rα₁ and IL-13Rα₂ is increased 3.5-fold and decreased 55%, respectively. (D) RT-PCR analyses of IL-4Rα mRNA expression in Beas2B cells exposed to control or alcohol (60 mM). No significant differences were observed between groups. 18S rRNA was used as a control for RNA input. Images are representative of 3 animals per group (immuno-histochemistry) or 3 independent experiments (RT-PCR). Bar = 250 μm (panels i and ii) and 50 μm (panels iii and iv). *^, ** p < 0.05 compared to Ctrl for each group.

Figure 4. Stimulation of STAT6 phosphorylation is amplified in cells exposed to alcohol

(A) Western blot analyses of STAT6 phosphorylation in Beas2B cells exposed to control (Ctrl) or alcohol (EtOH; 60 mM)) for 5 days. With 12 hours incubation remaining, some cells were exposed to LPS (+ LPS; 1 μg/ml). For the last 30 minutes, IL-13 (+ IL13; 10 ng/ml) was added to some cells to induce STAT6 phosphorylation. Top panels represent phospho-STAT6 blots of nuclear extracts (~100 kD band is marked by the arrow) and bottom panels represent STAT6 blots of cytoplasmic extracts. Alcohol alone does not lead to phosphorylation of STAT6; however, STAT6 phosphorylation is induced upon addition of IL-13. (B) Quantitation of phospho-STAT6 Western blots. STAT6 phosphorylation is amplified 2-fold in cells exposed to alcohol + LPS + IL-13 compared to controls + LPS + IL-13. * p < 0.05.

Figure 5. Chronic alcohol exposure to allograft donors modulates expression of components of pro-IL-13 signaling in the recipient

(A) Immuno-staining for α-SMA in untransplanted airways from control- and alcohol-fed donor rats. α-SMA staining is unaltered in airways from control- (panels i and iii) vs. alcohol-fed donors (panels ii and iv). (B) RT-PCR analyses of mRNA expression in untransplanted airways and 21-day allografts originating from control- or alcohol-fed donors. (C) Quantitation of RT-PCR analyses in untransplanted airways and 21-day allografts. IL-13 expression is increased in 21-day allografts (Ctrl vs. Ctrl HTT). IL-13 expression is induced in allografts originating from alcohol-fed donors (EtOH vs. EtOH HTT). IL-13Rα₁ expression is increased in untransplanted airways from alcohol-fed rats (Ctrl vs. EtOH). Upon transplantation (Ctrl HTT and EtOH HTT), expression of IL-13Rα₁ is increased. In contrast, IL-13Rα₂ expression is decreased in untransplanted airways from alcohol-fed rats (Ctrl vs. EtOH). Upon transplantation, mRNA levels of IL-13Rα₂ in allografts from alcohol-fed donors remained lower compared to allografts from control-fed donor rats. 18S rRNA was used as a control for RNA input. Immuno-staining and RT-PCR images are representative of 3 independent experiments. Bar = 250 μm (panels i and ii) and 50 μm (panels iii and iv). *^,**^,*** p < 0.05 compared to Ctrl for each gene.