Involvement of Igf1r in Bronchiolar Epithelial Regeneration: Role during Repair Kinetics after Selective Club Cell Ablation

<div><p>Regeneration of lung epithelium is vital for maintaining airway function and integrity. An imbalance between epithelial damage and repair is at the basis of numerous chronic lung diseases such as asthma, COPD, pulmonary fibrosis and lung cancer. IGF (Insulin-like Growth Factors) signaling has been associated with most of these respiratory pathologies, although their mechanisms of action in this tissue remain poorly understood. Expression profiles analyses of IGF system genes performed in mouse lung support their functional implication in pulmonary ontogeny. Immuno-localization revealed high expression levels of Igf1r (Insulin-like Growth Factor 1 Receptor) in lung epithelial cells, alveolar macrophages and smooth muscle. To further understand the role of Igf1r in pulmonary homeostasis, two distinct lung epithelial-specific <i>Igf1r</i> mutant mice were generated and studied. The lack of <i>Igf1r</i> disturbed airway epithelial differentiation in adult mice, and revealed enhanced proliferation and altered morphology in distal airway club cells. During recovery after naphthalene-induced club cell injury, the kinetics of terminal bronchiolar epithelium regeneration was hindered in <i>Igf1r</i> mutants, revealing increased proliferation and delayed differentiation of club and ciliated cells. Amid airway restoration, lungs of <i>Igf1r</i> deficient mice showed increased levels of <i>Igf1</i>, <i>Insr</i>, <i>Igfbp3</i> and epithelial precursor markers, reduced amounts of Scgb1a1 protein, and alterations in IGF signaling mediators. These results support the role of Igf1r in controlling the kinetics of cell proliferation and differentiation during pulmonary airway epithelial regeneration after injury.</p></div

mRNA expression and signaling mediator levels of epithelial cell markers and IGF system genes in lungs of <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> mutant mice during regeneration after naphthalene injury.

<p><b>(A)</b> mRNA expression levels of IGF system (<i>Igf1r</i>, <i>Igf1</i>, <i>Insr</i> and <i>Igfb3</i>), bronchiolar epithelium markers (<i>Scgb1a1</i>, <i>Cyp2f2</i> and <i>Sftpc</i>) and differentiation regulators (<i>Nkx2-1</i>, <i>Sox2</i>, <i>Notch3</i> and <i>Yap1</i>) in 3dN total lung extracts. Note that the reduced mRNA levels in <i>Igf1r</i> generate increased mRNA in <i>Insr</i>, <i>Igfbp3</i> and in epithelial precursor-related genes. <b>(B)</b> mRNA expression levels of <i>Igf1r</i>, and <i>Igf1</i> and <i>Nkx2-1</i>, in 7dN lungs. <i>Igf1</i> and <i>Nkx2-1</i> were the unique genes found with significant changes at this stage. <b>(C-D)</b> Representative Western blots and their graphical representations after quantification by densitometry for expression of Scgb1a1 (C) and phosphorylation and total levels of IGF signaling mediators (D), including phosphor-(p)-AKT and total Akt, pp38 and total p38, pERK1/2 and total ERK1/2, as well as pJNK/SAPK and total JNK/SAPK, using total lung extracts at 3dN and 7dN stages. Graphs represent blot band densitometric measurements after total protein loading normalization, using either Coomasie staining, or total content of each protein. Note the decreased levels of pERK and the increased levels of total JNK in mutant lungs at 7dN. Numbers in bars indicate number of mice analyzed. <i>+/+</i>, <i>Igf1r</i>^<i>fl/fl</i> and <i>Cre</i>/+, <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i>genotypes. Values in graphs show mean ± SEM. *, p<0.05; **, p<0.01.</p

Increased mRNA expression of IGF signaling genes after the naphthalene challenge.

<p>qRT-PCR analysis of <i>Igf1r</i> (A), <i>Igf1</i> (B) and <i>Insr</i> (C) mRNA levels before (NT), and three (3dN) and seven (7dN) days after naphthalene administration. Numbers in bars indicate mice analyzed. Values in graphs show mean ± SEM. Data are normalized to 18S RNA expression. RQ: relative quantity. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001 (Kruskal-Wallis test).</p

Specific <i>Igf1r</i> gene deletion and reduced expression in lungs of adult <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> double transgenic mice.

<p><b>(A)</b> PCR assays to determine the presence of the deleted allele (Δ) using F3/R1 primers on genomic DNA obtained from different organs of <i>Igf1r</i>^<i>fl/fl</i>(<i>+/+</i>) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> (<i>Cre/+</i>) mice. The 491 bp fragment of the deleted form <i>(Δ</i>) is present in lungs of <i>Cre/+</i> animals in detriment of the 1300 bp floxed <i>(fl)</i> allele (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166388#pone.0166388.s002" target="_blank">S1 Fig</a>, panels C-D), but absent in all other tissues tested, independently of the genotype. Ki, kidney; Li, liver; Lu, lung; Sp, spleen. <b>(B)</b><i>Igf1r</i> mRNA relative expression levels analyzed by qRT-PCR in lungs of <i>Igf1r</i>^<i>fl/fl</i> (+/+) (n = 5) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> (<i>Cre/+</i>) (n = 3) mice.<b>(C-F')</b> Immuno-staining for Igf1r in lungs of <i>Igf1r</i>^<i>fl/fl</i> (+/+) (C, C', E, E’) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> (<i>Cre/+</i>) (D, D', F, F'). Sections were co-stained either with Scgb1a1 to identify club cells in terminal bronchioles (C’-D’, orange/red), or with Pre-Sftpc to identify AEC2 in alveoli (E’-F’, orange/red). Note the lack of Igf1r green staining in both terminal bronchioles and AEC2 epithelial cells on <i>Cre/+</i> samples (white arrows in D and F, respectively), which do not co-stain with their specific markers, Scgb1a1 and PreSftpc, respectively (red arrows in D’ and F’). Remnant green staining in epithelial cell types could be due to either epithelial cells nonspecific autofluorescence or cells with undeleted <i>Igf1r</i> gene (mosaic deletion). Note the altered morphology and interruptions (white arrowheads in D-D’) in the epithelium of terminal bronchioles in the mutants (D-D’), compared to control mice (C-C’). al, alveolus; tb, terminal bronchiole. Scale bar in F': 50 μm in C-D'; 17 μm in E-F’.<b>(G)</b> Quantification of Igf1r relative fluorescence in bronchiolar and alveolar type 2 epithelial cells (n = 4 per genotype).<i>+/+</i>, <i>Igf1r</i>^<i>fl/fl</i> and <i>Cre</i>/+, <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i>genotypes. Values in graphs show mean ± SEM. *, p<0.05.</p

Histopathological changes in terminal bronchioles of <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> mutant mice during regeneration after naphthalene challenge.

<p><b>(A-D)</b> Representative H&E staining of bronchiole sections at three (3dN) (A-B) and seven (7dN) (C-D) days after the naphthalene treatment analyzed in <i>Igf1r</i>^<i>fl/fl</i> (<i>+/+</i>) (A, C) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i>(<i>Cre</i>/+) (B, D) mice. Red arrows in C point to club cells. Note the increased proportion of bronchiolar epithelial cells with ellipsoid nuclei protruding in the bronchiolar lumen (green arrows) and more abundant interruptions in epithelial continuity (black arrowheads) in double transgenic mutant mice (B, D). al, alveolus; tb, terminal bronchiole. Scale bars: 50 μm. <b>(E-G)</b> Quantification of bronchiolar epithelial alterations (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166388#pone.0166388.s004" target="_blank">S3 Fig</a> for representative images). Graphics represent changes in bronchiolar cell height (E), cell density (F), and interruptions (G) between genotypes in non-treated (NT), and at three (3dN), seven (7dN), fourteen (14dN) and twenty-four (24dN) days after the naphthalene treatment. <i>+/+</i>, <i>Igf1r</i>^<i>fl/fl</i> and <i>Cre</i>/+, <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i>genotypes. Numbers in bars indicate mice analyzed. Values in graphs show mean ± SEM. The statistical significance is indicated by specified symbols as follows: *, <i>+/Crevs</i>. <i>+/+</i> in same condition; @, <i>vs</i>. NT of same genotype; #, <i>vs</i>. 3dN of same genotype; $, <i>vs</i>. 7dN of same genotype; one symbol, <i>p</i><0.05; two symbols, <i>p</i><0.01; three symbols, <i>p</i><0.001.</p

Alterations in histology and cell proliferation in lungs of <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> mutant mice.

<p><b>(A-D)</b> Representative H&E staining of lung alveoli (A-B) and terminal bronchioles (C-D) sections obtained from <i>Igf1r</i>^<i>fl/fl</i> (<i>+/+</i>) (A, C) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i>(<i>Cre</i>/+) (B, D) mice. Note that in contrast to the normal alveolar parenchyma found in double transgenic mice (B), their bronchiolar epithelium shows histological alterations (D), including: lack of protruding club cells cupules (abundant in controls; red arrow in C), epithelial flattening (see shortening in red segments), lower cell density, abundance of interruptions (black arrowheads), presence of aberrant ellipsoid nuclei (green arrows) and big pale rounded cells (red arrowhead). al, alveolus; tb, terminal bronchiole. <i>+/+</i>, <i>Igf1r</i>^<i>fl/fl</i> and <i>Cre</i>/+, <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i>genotypes. Scale bars: 50 μm. <b>(E)</b> Graphical representations after quantification of bronchiolar epithelium alterations (cell height, cell density, and interruptions in epithelium continuity) in both genotypes (n = 4). <b>(F-I)</b> Representative images of Ki67 immuno-staining in brown (brown arrows) in alveolar parenchyma (F-G) and terminal bronchioles (H-I) of <i>Igf1r</i>^<i>fl/fl</i> (<i>+/+</i>) (F and H) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> (<i>Cre</i>/+) (G and I) mice. Note the increased presence of Ki67⁺ cells in the bronchiolar epithelium of <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> mutant mice. <b>(J)</b> Graphical representation of Ki67⁺ based proliferation rates (n = 4). <b>(K)</b> mRNA expression levels of genes analyzed by qRT-PCR in total lung homogenates from <i>Igf1r</i>^<i>fl/fl</i> (<i>+/+</i>) (n = 5) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> (<i>Cre</i>/+) (n = 3) three month old mice. Values in graphs show mean ± SEM. * p<0.05.</p

Increased cell proliferation rates in terminal bronchioles of <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> mice at different stages during repair after naphthalene challenge.

<p><b>(A-B)</b> Representative images of Ki67 immunostaining (brown arrows) in terminal bronchioles of <i>Igf1r</i>^<i>fl/fl</i> (+/+) (A) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> (<i>Cre</i>/+) (B) lungs at 3dN. Note the increased presence of Ki67⁺ in the epithelium of <i>Cre</i>/+ mutant mice with respect to <i>+/+</i> controls. <b>(C)</b> Quantification of Ki67-based proliferation rates in both genotypes (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166388#pone.0166388.s008" target="_blank">S7 Fig</a> for representative images). Numbers in bars indicate mice analyzed. <b>(D-G)</b> Identification of proliferating cells by anti-BrdU immunostaining (green labeled nuclei) counterstained in red with an anti-Scgb1a1 antibody to mark club cells, allowing for differentiation of three groups of cells: total BrdU⁺ cells in the bronchiolar epithelium (including Scgb1a1⁺ cells) (green arrows), BrdU⁺/Scgb1a1⁺ double labeled cells (red arrows) and BrdU⁺ cells under the epithelial basal membrane (blue arrows) at 3dN (D-E) and 7dN (F-G) in control (<i>+/+</i>) (D, F) and mutant (<i>Cre/+</i>) (E, G) lungs. al, alveolus; tb, terminal bronchiole. Scale bars: 50 μm in A-B; 25 μm in D-G. <b>(H)</b> Quantification of BrdU⁺ cells in terminal bronchioles of control (<i>+/+</i>) and mutant (<i>Cre/+</i>) mice. The graph represents the quantification of total BrdU+ cells found in the epithelium (green bars), BrdU⁺/Scgb1a1⁺ club epithelial cells (red/pink bars) and in blue bars BrdU+ cells under the basal membrane (up to 12 μm distance) in non-treated (NT), and at three (3dN), seven (7dN) and fourteen (14dN) days after naphthalene treatment (red/pink bars) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166388#pone.0166388.s009" target="_blank">S8 Fig</a> for representative images). Values in graphs show mean ± SEM. The statistical significance between stages is indicated by specified symbols as follows: *, <i>+/Crevs</i>. <i>+/+</i> in same condition; @, <i>vs</i>. NT of same genotype; #, <i>vs</i>. 3dN of same genotype; $, <i>vs</i>. 24dN of same genotype; one symbol, <i>p</i><0.05; two symbols, <i>p</i><0.01; three symbols, <i>p</i><0.001.</p

Delayed regeneration of club and ciliated cells in <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> terminal bronchioles after naphthalene challenge.

<p><b>(A-D)</b> Representative images of immuno-staining to identify club/Scgb1a1⁺ cells (in red) and ciliated/GluTub⁺ (in green) in lungs of <i>Igf1r</i>^<i>fl/fl</i> (+/+) (A, C) and <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i> (<i>Cre/+</i>) (B, D) at 7dN (A-B) and 14dN (C-D) days after naphthalene treatment. Note the lower proportion of club (red arrows) and ciliated cells (green arrows), and the increased presence of non-labeled epithelial cells (blue arrows) in <i>Cre/+</i> mutants. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166388#pone.0166388.s006" target="_blank">S5 Fig</a> for representative images). al, alveolus; tb, terminal bronchiole. Scale bar in D: 50 μm. <b>(E)</b> Quantification of bronchiolar epithelial cell types labeled in the bronchiolar epithelium following the above-mentioned criteria. <i>+/+</i>, <i>Igf1r</i>^<i>fl/fl</i> and <i>Cre</i>/+, <i>Nkx2-1-Cre; Igf1r</i>^<i>fl/fl</i>genotypes (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166388#pone.0166388.s006" target="_blank">S5 Fig</a> for representative images). Values in graphs show mean ± SEM. Unlabel, unlabeled cells with either Scgb1a1 or GluTub. The statistical significance between stages is indicated by specified symbols as follows: *, <i>+/Crevs</i>. <i>+/+</i> in same condition; @, <i>vs</i>. NT of same genotype; #, <i>vs</i>. 3dN of same genotype; $, <i>vs</i>. 7dN of same genotype; &, <i>vs</i>. 14dN of same genotype; one symbol, <i>p</i><0.05; two symbols, <i>p</i><0.01; three symbols, <i>p</i><0.001.</p