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

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

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 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

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

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

Histology of the joints in WT and TNF^ΔARE mice.

<p>H&E staining of the ankle joints of <b>(A)</b> air-exposed WT mice, <b>(B)</b> CS-exposed WT mice, <b>(C)</b> air-exposed TNF^ΔARE mice and <b>(D)</b> CS-exposed TNF^ΔARE mice. <b>(E)</b> Histological score of inflammation in the joints of WT and TNF^ΔARE mice. Histological inflammation is increased in TNF^ΔARE mice compared to WT mice (p < 0,005). Data are expressed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

Mean TNF-α levels in serum of WT and TNF^ΔARE mice.

<p>Protein levels of TNF-α in serum of WT and TNF^ΔARE mice throughout the 4 weeks air- or CS-exposure. As expected, TNF-α was increased in TNF^ΔARE mice compared to WT mice (p < 0,005).</p