Characterization of a lung epithelium specific E-cadherin knock-out model: Implications for obstructive lung pathology

S Post, I H Heijink, L Hesse, H K Koo, F Shaheen, M Fouadi, V N S Kuchibhotla, B N Lambrecht, A J M Van Oosterhout, T L Hackett, M C Nawijn, S Post, I H Heijink, L Hesse, H K Koo, F Shaheen, M Fouadi, V N S Kuchibhotla, B N Lambrecht, A J M Van Oosterhout, T L Hackett, M C Nawijn

Abstract

The airway epithelium regulates responses to aeroallergens, acting as a physical and immunological barrier. In asthma, epithelial barrier function and the expression of adherens junction protein E-cadherin is compromised, but it is unknown whether this is cause or consequence of the disease. We hypothesized that airway epithelial loss of E-cadherin is a critical step in the development of manifestations of asthma. We generated a transgenic mouse model with conditional loss of E-cadherin in lung epithelial cells at birth and onwards. We observed normal lung development at the time of birth in mice lacking E-cadherin in the lung epithelium. However, E-cadherin deficiency led to progressive epithelial damage in mice growing into adulthood, as evidenced by airway epithelial denudation, decreased zonula occludens (ZO)-1 expression, loss of ciliated cells, and enlarged alveolar spaces. In addition, spontaneous goblet cell metaplasia with mucus production was observed. These epithelial changes were accompanied by elevated levels of the epithelial-derived chemokine CCL17, infiltration of eosinophils and dendritic cells, and mucus production. In conclusion, loss of E-cadherin induces features in the lung reminiscent of those observed in asthma, indicating that the disruption of E-cadherin-mediated cell-cell contacts may play a key role in the development of asthma manifestations.

Conflict of interest statement

This study was funded by unrestricted research grants from the Dutch Lung Foundation (Longfonds; 3.2.07.019), Stichting Astma Bestrijding (2014/008), European Respiratory Society (LTRF-118-2012) and the Canadian Institutes for Health Research (260046). We have no non-financial competing interests.

Figures

Figure 1
Figure 1
E-cadherin expression in the lungs of E-cadherin knockout (Cdh1fl/fl Cre+) and wild type (Cdh1fl/fl Cre−) mice (n = 5–7 per group). (A) E-cadherin staining of airway epithelium at day (D)0 (I,IV), week (W) 4 (II,V) and W10 (III,VI) of Cdh1fl/fl Cre−(I-III)/Cre+ (IV-VI) mice. Red arrows indicate epithelial denudation areas. Scale bars: 10 μm. Percentage of E-cadherin positive cell numbers as analyzed by Image-Pro Plus at (B) D0, (C) W4 and (D) W10. mRNA expression of E-cadherin (cdh1) in lung homogenates at (E) W4 and (F) W10. E-cadherin levels were related to the housekeeping genes hprt1 and pgk1 and expressed as 2−ΔCt. Medians are indicated. *p < 0.05, **p < 0.01 and ***p < 0.001 as assessed by the Mann Whitney U test.
Figure 2
Figure 2
Characterization of airway epithelium in E-cadherin knockout (Cdh1fl/fl Cre+) and wild type (Cdh1fl/fl Cre−) mice. Analysis of percent total cells in the epithelium of Cdh1fl/fl Cre−/Cre+ mice at (A) day (D)0, (B) W4 and (C) W10, where the total cell count per 500 µm basement membrane (5–25 counts per lung fragment) per mouse is presented as percentage of the group average. Medians are indicated. (D) Electron microscopy images at W4, magnifications are as indicated. White arrows indicate epithelial denudation areas. (E) Hematoxylin staining in lung tissue of Cdh1fl/fl Cre−/Cre+ mice at W8, magnifications are as indicated. *p < 0.05, **p < 0.01 and ***p < 0.001 between the Cdh1fl/fl Cre+ and Cdh1fl/fl Cre− mice (n = 5–7 per group) as assessed by the Mann Whitney U test.
Figure 3
Figure 3
Characterization of alveoli in E-cadherin knockout (Cdh1fl/fl Cre+) and wild type (Cdh1fl/fl Cre−) mice. (A) E-cadherin staining of alveoli at day (D)0 (I,IV), week (W)4 (II,V) and W10 (III,VI) of Cdh1fl/fl Cre− (I-III)/Cre+ (IV-VI) mice. Scale bars: 10 μm. Measurement of mean linear intercept (Lm) in the lung tissue at (B) D0, (C) W4 and (D) W10. (E) Hematoxylin staining in lung tissue of Cdh1fl/fl Cre−/Cre+ mice at D0, W4 and W10. Scale bars, 100 μm. An average Lm was calculated from 3 randomly selected regions within each lung. Lm = Number of lines × Length of test line/Number of intersections. A greater Lm value therefore indicates increased air-space size.Medians are indicated. *p < 0.05, **p < 0.001 and ***p < 0.0001 between the Cdh1fl/fl Cre+ and Cdh1fl/fl Cre− mice (n = 5–7 per group) as assessed by the Mann Whitney U test.
Figure 4
Figure 4
Loss of ciliated cells and increased mucus production in E-cadherin knockout (Cdh1fl/fl Cre+) and wild type (Cdh1fl/fl Cre−) mice. Analysis of percent ciliated cells at (A) day (D)0, (B) week (W)4 and (C) W10, where the ciliated cell count per mouse was calculated per 500 µm basement membrane (5–25 counts per lung fragment) as percentage of the group average. (D) Electron microscopy images at Week W6. Red arrow indicates loss of ciliated cells. Measurement of percentage alcian blue staining in the airways of Cdh1fl/fl Cre−/Cre+ mice at E) W4, (F) W8 and (G) W10, (H) Representative image of alcian blue staining of Cdh1fl/fl Cre−/Cre+ mice at W8. Scale bars, 10 μm. (I) Percentage of alpha-smooth muscle actin (α-SMA) positive cell numbers as analyzed by Image-Pro Plus in the airways of Cdh1fl/fl Cre−/Cre+ mice at W10. Medians are indicated. *p < 0.05 and **p < p0.01 between the Cdh1fl/fl Cre+ and Cdh1fl/fl Cre− mice (n = 5–7 per group) as assessed by the Mann Whitney U test.
Figure 5
Figure 5
Cytokine responses in E-cadherin knockout (Cdh1fl/fl /Cre+) and wild type (Cdh1fl/fl Cre−) mice.Enzyme-linked immunosorbent assay (ELISA) analysis for (A) Chemokine (C-C motif) ligand 17 (CCL17), (B) Thymic stromal lymphopoietin (TSLP), (C) C-C motif chemokine 11 (CCL11) and (D) Granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in whole lung homogenates at week (W)4. Medians are indicated. **p < 0.01 between the Cdh1fl/fl Cre+ and Cdh1fl/fl Cre− mice (n = 5–7 per group) as assessed by the Mann Whitney U test.
Figure 6
Figure 6
Inflammatory responses in E-cadherin knockout (Cdh1fl/fl Cre+) mice) and wild type (Cdh1fl/fl Cre−) mice. Flow cytometry analysis for inflammatory cells in whole lungs of Cdh1fl/fl Cre−/Cre+ mice at (A) week (W)2 and (E) W4. Dendritic cell (DC) populations at (B) W2 and F) W4 and DC sub-populations (CD103+ conventional (c)DCs, CD11b + DCs and monocyte-derived (mo)DCs) at (C) W2 and (G) W4. Alveolar macrophages at (D) 2 W and (H) 4 W. Medians are indicated. *p < 0.05, **p < 0.01 or p value is as indicated between the Cdh1fl/fl Cre+ and Cdh1fl/fl Cre− mice (n = 5–7 per group) as assessed by the Mann Whitney U test.

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