Intracellular cytokine production in the pulmonary ILC population.

<p>Several signature cytokines in pulmonary ILC (gated as CD45⁺, Lin^- CD127⁺ cells) were determined on single cell suspensions of digested human lung (n = 8). Since NK cells could contaminate the non-toxic ILC1 subset, CD56⁺ cells were excluded to investigate the IFN-γ production. For the production of these cytokines, lung cells were first stimulated for 15 hours with PMA/ionomycin (+ transport inhibitors). <b>A,</b> IFN-γ production in ILC. <b>B,</b> Production of IL-5 in the pulmonary ILC population. <b>C,</b> IL-17 production in ILC. <b>D,</b> ILC production of IL-22. <b>E,</b> Production of GM-CSF in the ILC population. The bottom panels represent the isotype controls of the specific cytokine staining. <b>F,</b> Frequency of IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF positive cells within the ILC (CD45⁺, Lin^- CD127⁺) population (n = 8, mean ± SEM).</p

Overview of innate lymphoid cell subsets in human lung tissue.

<p>The presence of CD45⁺, Lin^- (i.e. CD3, CD19, CD11c, CD11b) and CD127⁺ ILC in pulmonary tissue was demonstrated. These ILC were further subdivided in a CD56^- IL12Rβ2⁺ ILC1 subset, CRTH2⁺ ILC2 subset, CD117⁺ NKp44⁺ (NCR⁺) ILC3 subset and CD117⁺ NKp44^- (NCR^-) ILC3 subset. Further, expression of signature transcription factors (i.e. T-bet, GATA-3 and RORγT) within the specific ILC subset and cytokine production (i.e. IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF) within the pulmonary ILC population was demonstrated.</p

ILC subsets in control subjects versus patients with COPD.

<p><b>A,</b> The frequency of ILC1 (CD45⁺, Lin^-, CD127⁺, CD56^-, IL12Rβ2⁺), ILC2 (CD45⁺, Lin^-, CD127⁺, CRTH2⁺), NCR⁺ ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺, NKp44⁺) and NCR^- ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺, NKp44^-) in digested human lung from control (n = 5) and COPD patients (n = 11) was determined by flow cytometry. ILC numbers were expressed as percentages (%) of the CD45⁺ population (mean ± SEM). <b>B,</b> Pie chart of the relative abundance of ILC1, ILC2, NCR⁺ ILC3 and NCR^- ILC3 subsets in control subjects and patients with COPD.</p

Intracellular staining of transcription factors in pulmonary ILC subsets.

<p>The developmental transcription factors were determined in the specific ILC subsets on single cell suspensions of digested human lung. <b>A,</b> Expression of T-bet (black line) versus isotype control (grey line) in the ILC1 (CD45⁺, Lin^- CD127⁺, CD56^-, CRTH2^-, CD117^-), ILC2 (CD45⁺, Lin^-, CD127⁺, CRTH2⁺), ILC3 (CD45⁺, Lin^-, CD127⁺, CD117⁺) population. <b>B,</b> GATA-3 expression (black line) versus isotype control (grey line) in the different ILC subsets. <b>C,</b> Expression of RORγT (black line) versus isotype control (grey line) in ILC1, ILC2 and ILC3 population.</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

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 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

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

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