Airway surface dehydration aggravates cigarette smoke-induced hallmarks of COPD in mice

Introduction: Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown. Objective: We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the β-subunit of the epithelial Na+ channel (βENaC). Methods: βENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured. Results: Airway surface dehydration in βENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in βENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements. Conclusions: We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD

Overexpression of βENaC and reduced airway surface liquid height in βENaC-Tg mice.

<p>Immunolocalization of βENaC in airways from WT and βENaC-Tg mice. <b>(A)</b> Representative images of βENaC immunostaining of lung sections from WT and βENaC-Tg mice that were exposed to air or CS for 8 weeks. n = 5 per group. Dysregulation of steady state airway surface liquid (ASL) height on airway epithelia from βENaC-Tg mice under thin film conditions. Representative confocal images <b>(B)</b> and summary of measurements of airway surface liquid height <b>(C)</b> at t = 0, 2, 4, 8 and 24h after mucosal addition of 20 μl of PBS containing Rhodamine dextran to primary tracheal epithelial cultures from βENaC-Tg mice and WT littermates. Scale bar, 7 μm. n = 4 experiments per group. *p<0.001 compared to βENaC-Tg; ^§p<0.001 for t = 0h compared to all other time points within the same genotype; ^†p<0.05 for t = 24h wild-type compared to t = 2h wild-type; ^‡p<0.005 for t = 24h wild-type compared to t = 4h and 8h wild-type.</p

Cigarette smoke-induced mucin expression is increased in βENaC-Tg mice.

<p>mRNA expression of Muc5ac <b>(A)</b> and Muc5b <b>(B)</b> in total lung tissue upon 4 weeks of air or CS exposure. mRNA expression data were normalized for 3 reference genes (Hprt1, Gapdh, Tfrc). n = 6/group. *p<0.05, **p<0.01, ***p<0.001.</p

Cigarette smoke-induced alveolar destruction is increased in βENaC-Tg mice.

<p><b>(A)</b> Mean linear intercept (Lm) upon 4 weeks of air or CS exposure. n = 7-8/group. Representative image of WT mice: air-exposed <b>(B)</b> and CS-exposed <b>(C)</b>. Destructive index (DI) upon 4 weeks of air or CS exposure <b>(D)</b>. n = 7-8/group. Representative image of βENaC-Tg mice: air-exposed <b>(E)</b> and CS-exposed <b>(F)</b>. mRNA expression of Mmp12 in total lung upon 4 weeks of air- or CS exposure <b>(G)</b>. Normalized for 3 reference genes (Hprt1, Gapdh, and Tfrc). n = 6/group. *p<0.05, **p<0.01, ***p<0.001.</p

mRNA expression and protein levels of chemokines upon air or CS exposure.

<p>mRNA expression of Cxcl1 in total lung tissue upon 4 weeks <b>(A)</b> and 8 weeks <b>(C)</b> air or CS exposure. mRNA expression of Ccl20 in total lung tissue upon 4 weeks <b>(B)</b> and 8 weeks <b>(D)</b> air or CS exposure. mRNA expression data were normalized for 3 reference genes (Hprt1, Gapdh, Tfrc). n = 6-8/group. Protein levels of Cxcl1 in BAL fluid upon 4 weeks <b>(E)</b> and 8 weeks <b>(G)</b> air or CS exposure. Protein levels of Ccl20 in BAL fluid upon 4 weeks <b>(F)</b> and 8 weeks <b>(H)</b> air or CS exposure. Protein levels were measured by ELISA. n = 8-11/group. *p<0.05, **p<0.01, ***p<0.001.</p

Cigarette smoke-induced pulmonary inflammation is increased in βENaC-Tg mice.

<p><b>(A)</b> Total inflammatory cell count in BAL upon 4 weeks of air or CS exposure. Quantification of macrophages <b>(B)</b>, neutrophils <b>(C)</b> and lymphocytes <b>(D)</b> in BAL upon 4 weeks of air or CS exposure. n = 7-8/group. <b>(E)</b> Quantification of macrophages in total lung after 4 weeks of CS exposure. n = 7-8/group. Quantification of dendritic cells <b>(F)</b>, CD4+ T-lymphocytes <b>(G)</b> and CD8+ T-lymphocytes <b>(H)</b> in BAL upon 8 weeks of air or CS exposure. n = 8-11/group. *p<0.05, **p<0.01, ***p<0.001.</p

Cigarette smoke-induced lymphoid follicle formation in βENaC-tg, but not in WT mice after 8 weeks of CS exposure.

<p><b>(A)</b> Quantification of lymphoid follicles normalized for the area of parenchyma (mm²) upon 8 weeks of air or CS exposure. n = 8-11/group. <b>(B–C)</b> Representative images of lymphoid follicles found in CS-exposed βENaC-Tg mice. *p<0.05, **p<0.01, ***p<0.001.</p

Goblet cell metaplasia and mucus secretion upon air or CS exposure.

<p><b>(A)</b> Goblet cell count upon 4 weeks of air or CS exposure. n = 8/group. Representative images of goblet cells in airways of CS-exposed WT mice <b>(B)</b> and CS-exposed βENaC-Tg mice <b>(C)</b> upon 4 weeks of CS exposure. Arrows indicate goblet cells. <b>(D)</b> Quantification of PAS+ mucus content in lumen of airways of non-lavaged mice upon 4 weeks of air or CS exposure n = 3/group. Representative image of PAS+ mucus content in airways of CS-exposed WT mice <b>(E)</b> and CS-exposed βENaC-Tg mice <b>(F)</b>. *p<0.05, **p<0.01, ***p<0.001.</p

Effect of CS exposure on lung function in WT and βENaC-Tg mice.

<p>Lung function was determined in WT and βENaC-Tg mice after exposure to air or CS for 4 weeks. <b>(A)</b> Resistance (R) of the entire compartment (airways, tissue and chest wall). <b>(B)</b> Tissue damping (G), related to tissue resistance. <b>(C)</b> Tissue elasticity (H). <b>(D)</b> Static compliance (C_stat). <b>(E)</b> Dynamic compliance (C_dyn). <b>(F)</b> Total lung capacity (TLC). *p<0.05, **p<0.01, ***p<0.001.</p