Lung inflammation and interstitial fibrosis by targeted alveolar epithelial type I cell death

Abstract

Introduction: The pathogenesis of chronic lung diseases is multifaceted with a major role of recurrent micro-injuries of the epithelium. While several reports clearly indicated a prominent role for surfactant-producing alveolar epithelial type 2 (AT2) cells, the contribution of gas exchange-permissive alveolar epithelial type 1 (AT1) cells has not been addressed yet. Here, we investigated whether repeated injury of AT1 cells leads to inflammation and interstitial fibrosis.

Methods: We chose an inducible model of AT1 cell depletion following local diphtheria toxin (DT) administration using an iDTR flox/flox (idTR^fl/fl) X Aquaporin 5CRE (Aqp5^CRE) transgenic mouse strain.

Results: We investigated repeated doses and intervals of DT to induce cell death of AT1 cells causing inflammation and interstitial fibrosis. We found that repeated DT administrations at 1ng in iDTR^fl/fl X Aqp5^CRE mice cause AT1 cell death leading to inflammation, increased tissue repair markers and interstitial pulmonary fibrosis.

Discussion: Together, we demonstrate that depletion of AT1 cells using repeated injury represents a novel approach to investigate chronic lung inflammatory diseases and to identify new therapeutic targets.

Keywords: AT1 cell depletion; cell death; iDTR; lung injury; sterile inflammation.

Figure 1

Generation and validation of iDTR^fl/fl x Aqp5^CRE mice. A CRE recombinase cassette was placed under control of the aquaporin5 (AQP5) promoter in a knockin mouse strain for ATI cell-specific targeting and crossed with iDTR floxROSA stop mice. In the absence of CRE recombinase activity, a stop cassette impedes DTR expression. In AT1 cells, Aqp5 promoter activation induces CRE expression which removes the STOP cassette inserted upstream of the DTR gene and allows its transcription (A). iDTR^fl/fl x Aqp5 ^CRE mice were treated with NaCl for two consecutive days and lungs harvested 24 h after the last exposure and lung sections were stained with anti-podoplanin (Pdpn) and anti-prosurfactant protein C (ProSP-C) to visualize AT1 and AT2 cells, respectively (B). iDTR^fl/fl x Aqp5^CRE mice and control iDTR^fl/fl x Aqp5^WT mice were treated daily for two days with 1 ng of DT in the airways by oropharyngeal administration and harvested 24 h after the last administration (C). Lung immunofluorescence imaging suggests discrete alveolar loss in iDTRfl/fl x Aqp5CRE (D), with strong increases of cleaved caspase-3 (Casp-3) staining in AT1 cells (E, F). Mice were treated daily for three consecutive days with 10 ng of DT and harvested at day 7 (G). Representative flow cytometry plot (H), percentage (I) and total numbers (J) of AT1 cells recovered in the bronchoalveolar lavage fluid (BALF) gated as Pdpn⁺ and pancytokeratin (Pan CK)⁺. Each dot represents an individual sample; data are representative of 2 independent experiments, showed as mean ± SEM, *p < 0.05; **p < 0.01.

Figure 2

DT-induced AT1 cell ablation causes inflammatory cell recruitment and early lung inflammation at day 3. iDTR^fl/fl x Aqp5^CRE mice and control iDTR^fl/fl x Aqp5^WT mice were treated daily for three consecutive days with 10 ng of DT in the airways by oropharyngeal administration and harvested 24 h after the last administration. An additional iDTR^fl/fl x Aqp5^WT mice control group received saline (NaCl) administration (A). Cell free protein (B) and cell free-DNA (C) contents in the BALF were significantly increased as well as total cell counts (D), especially epithelial cells (E) and neutrophils (F) in the DT-treated iDTR^fl/fl x Aqp5^CRE group. Lung tissue was stained with hematoxylin and eosin (HE) (G) and lung inflammation and injured scored (H, I). Each dot represents an individual sample; data are representative of a single experiment, shown as mean ± SEM, *p < 0.05; **p < 0.01.

Figure 3

DT-induced AT1 cell ablation causes inflammatory cell recruitment at day 8. iDTR^fl/fl x Aqp5^CRE mice and control iDTR^fl/fl x Aqp5^WT mice were treated at days 0 and 2 with 10 ng of DT in the airways by oropharyngeal administration and harvested at day 8. An additional iDTR^fl/fl x Aqp5^CRE mice control group received saline administration (A). Significant body weight loss was observed in DT-treated iDTR^fl/fl x Aqp5^CRE mice starting at day 5 (B). Increased BALF cell-free self-DNA content in DT-treated iDTR^fl/fl x Aqp5^CRE (C). Representative flow cytometry plots (D), percentage (E) and total numbers (F) of BALF epithelial cells. Numbers of CD45⁺ cells in the BALF (G) and pie chart showing main immune cell populations (H). Increased neutrophils (I) and eosinophils (J) in DT-treated iDTR^fl/fl x Aqp5^CRE mice as well as elevated levels of remodeling factors MMP-9 (K) and TIMP1 (L) in the BALF. Each dot represents an individual sample; data are representative of 2 independent experiments, showed as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.01; ns, non-significant.

Figure 4

DT-induced AT1 cell ablation causes lung inflammation and early fibrosis at day 8. Mice were treated as in Figure 3A and lung tissue was stained with H&E (A) and scored for inflammation (B) and injury (C). Collagen deposition in the lungs was assessed by Red Sirius staining showing collagen fibers in purple (D) and collagen content measured by Sircol assay (E). Increased levels of remodeling factors MMP-9 (F) and TIMP1 (G) were found in the lungs. CD45⁺ cell numbers were increased in the lungs of DT-treated iDTR^fl/fl x Aqp5^CRE mice (H). Main immune cell populations are showed in a pie chart (I) and significant increase of both neutrophils (J) and alveolar macrophages (K) were observed. Each dot represents an individual sample; data are representative of 2 independent experiments, showed as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant.

Figure 5

Low dose DT-induced AT1 cell ablation causes inflammation at day 16. iDTR^fl/fl x Aqp5^CRE mice and control iDTR^fl/fl x Aqp5^WT mice were treated at days 0, 2 and 13 with low dose (1ng) of DT in the airways by oropharyngeal administration and harvested at day 16. An additional iDTR^fl/fl x Aqp5^CRE mice control group received saline administration (A). Significant body weight loss in DT-treated iDTR^fl/fl x Aqp5^CRE mice starting at day 6 with full recovery by day 12 (B). Increased BALF cell-free self-DNA content in DT-treated iDTR^fl/fl x Aqp5^CRE (C), together with increased percentage (D) and total numbers (E) of epithelial cells as well as CD45⁺ immune cells (F). Pie chart showing main BALF immune cells populations (G) and increased numbers of eosinophils, CD4⁺ and CD8⁺ T lymphocytes in DT-treated iDTR^fl/fl x Aqp5^CRE mice and neutrophils and B lymphocytes in a lesser extent (H). Each dot represents an individual sample; data are representative of 2 independent experiments, showed as mean ± SEM, *p < 0.05; ns, non-significant.

Figure 6

Low dose DT-induced AT1 cell ablation causes lung inflammation and fibrosis at day 16. Mice were treated as depicted in Figure 5A and lung tissue was stained with H&E (A) and scored for inflammation (B) and injury (C). Collagen deposition in the lungs was assessed by Red Sirius stain (D) and collagen content in the BALF measured by Sircol assay (E). Gene expression of fibrosis markers collagen (Col3a), fibronectin (Fn1) and Timp1 are increased in DT-treated iDTR^fl/fl x Aqp5^CRE mice (F), whereas interferon lambda2 (Ifnl2) expression is strongly decreased (G). Western blot assay showed increased STING expression in DT-treated iDTR^fl/fl x Aqp5^CRE mice (H, I). Each dot represents an individual sample; data are representative of 2 independent experiments, showed as mean ± SEM, *p < 0.05; **p < 0.01; ns, non-significant.