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. 2019 Apr 15;129(5):2107-2122.
doi: 10.1172/JCI125014.

Yap/Taz regulate alveolar regeneration and resolution of lung inflammation

Ryan LaCanna  1 , Daniela Liccardo  1 , Peggy Zhang  1 , Lauren Tragesser  1 , Yan Wang  2 , Tongtong Cao  1 , Harold A Chapman  3 , Edward E Morrisey  4 , Hao Shen  2 , Walter J Koch  1 , Beata Kosmider  5 , Marla R Wolfson  5 , Ying Tian  1
Affiliations

Yap/Taz regulate alveolar regeneration and resolution of lung inflammation

Ryan LaCanna et al. J Clin Invest. .

Abstract

Alveolar epithelium plays a pivotal role in protecting the lungs from inhaled infectious agents. Therefore, the regenerative capacity of the alveolar epithelium is critical for recovery from these insults in order to rebuild the epithelial barrier and restore pulmonary functions. Here, we show that sublethal infection of mice with Streptococcus pneumoniae, the most common pathogen of community-acquired pneumonia, led to exclusive damage in lung alveoli, followed by alveolar epithelial regeneration and resolution of lung inflammation. We show that surfactant protein C-expressing (SPC-expressing) alveolar epithelial type II cells (AECIIs) underwent proliferation and differentiation after infection, which contributed to the newly formed alveolar epithelium. This increase in AECII activities was correlated with increased nuclear expression of Yap and Taz, the mediators of the Hippo pathway. Mice that lacked Yap/Taz in AECIIs exhibited prolonged inflammatory responses in the lung and were delayed in alveolar epithelial regeneration during bacterial pneumonia. This impaired alveolar epithelial regeneration was paralleled by a failure to upregulate IκBa, the molecule that terminates NF-κB-mediated inflammatory responses. These results demonstrate that signals governing resolution of lung inflammation were altered in Yap/Taz mutant mice, which prevented the development of a proper regenerative niche, delaying repair and regeneration of alveolar epithelium during bacterial pneumonia.

Keywords: Adult stem cells; Bacterial infections; Pulmonology; Stem cells.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Alveolar epithelial injury and recovery in SpT4-infected mice.
Lung tissues were collected at 0, 2, 4, 7, and 14 dpi with SpT4. (A) Immunostaining on lung sections with antibodies to the type 4 capsule of SpT4, T1a, and pro-SPC (SPC). Cell apoptosis was measured by TUNEL staining. Cell nuclei were stained with DAPI (blue). Scale bars: 50 μm. (B) Mouse lungs were homogenized, and lung lysates were plated for quantitative culture of colonizing pneumococci. (C) Quantification of cell apoptosis by counting TUNEL+ cells on lung sections. (D) Lung cells were dissociated, and T1a+ cells were quantified as percentage of total CD45– cells by flow cytometry. (E) Quantification of the number of SPC+ cells on lung sections. n ≥ 10 randomly selected fields per animal (C and E); n = 3–9 per group (B–E). **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA.
Figure 2
Figure 2. Alveolar epithelial regeneration and Yap/Taz expression in mouse lungs during bacterial pneumonia.
(A) Schematic of experimental design (left) and confocal images of lung sections at 0 dpi and 4 dpi. AECIIs in DNA synthesis phase were detected using Click-iT EdU Alexa Fluor (green) and coimmunostaining with antibody against pro-SPC (SPC) (red). Cell nuclei were stained with DAPI (blue). (B) Quantification of EdU+SPC+ cells as percentage of total SPC+ cells analyzed (~2200 SPC+ cells per animal). (C) Confocal images of lung sections of SPC-CreERT2, Rosa26-mTmG mice at 0, 7, and 14 dpi. Mice were administrated with 3 doses of tamoxifen to label SPC+ AECIIs. Fourteen days after the last tamoxifen treatment, mice were infected with SpT4. AECII-to-AECI differentiation was visualized by coimmunostaining with antibodies against GFP (lineage-labeled AECIIs) and T1a (AECIs). Arrowheads point to regions double-positive for GFP and T1a. (D) Quantification of percentage of GFP+T1a+ area of total GFP+ area per field using ImageJ. (E) Flow cytometry analysis of dissociated lung cells showing the percentage of GFP+T1a+ cells of total T1a+ cells at indicated time points. (F) Confocal images of lung sections of SPC-CreERT2, Rosa26-mTmG mice. Immunostaining with antibodies against GFP (lineage-labeled AECIIs) and Yap and Taz. Cell nuclei were stained with DAPI (blue). (G) Western blot using lung tissue lysates at 0 dpi or purified AECIIs at 0 and 7 dpi, blotted with anti-YAP, anti-pYAP (Ser127), anti-Taz, anti-pTaz (S89), and anti–β-actin. Histograms showed average of total YAP or TAZ normalized to β-actin (loading control), together with average ratio of pYAP/YAP and pTAZ/TAZ. n ≥ 4 per group (B, D, E); n = 3 per group (G). *P < 0.05; **P < 0.01; ****P < 0.0001, 1-way ANOVA (B, D, E) and Student’s t test (G). Scale bars: 10 μm.
Figure 3
Figure 3. Phenotypes of Yap/Taz mutant lungs during bacterial pneumonia.
(A) Immunostaining on lung sections with nuclei labeled by DAPI (blue) and antibodies to T1a (red) or pro-SPC (SPC) (green). (B) Lung cells were dissociated and T1a+ cells were quantified as percentage of total CD45– cells by flow cytometry. SPC+ cells were quantified by counting the number of SPC+ cells per field (≥10 randomly selected fields per animal) (n = 3–8 per group). (C) Lung tissue sections were stained with Alcian blue and Nuclear Fast Red. Lung fibrotic lesions were quantified by measuring (D) Aschcroft score and (E) hydroxyproline assay (n = 3–4 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, 2-way ANOVA. Scale bars: 50 μm (A, C [bottom panel]); 500 μm (C [top panel]).
Figure 4
Figure 4. AECII proliferation and differentiation in Yap/Taz mutant lungs during bacterial pneumonia.
(A) Schematic of experimental design for studies shown in B–F. (B) Lung tissue sections from SPC-CreERT2, Rosa26-mTmG mouse were immunostained with DAPI (blue) and antibody against GFP (lineage-labeled AECIIs) (green), and colabeling with Click-iT EdU Alexa Fluor (red) and confocal images were taken. The percentages of GFP+EdU+ cells of total GFP+ cells per field were graphed (bottom panel). (C) Confocal image of lung section from SPC-CreERT2, Rosa26-mTmG mouse at 7 dpi with nuclei labeled by DAPI (blue) and antibodies against GFP (green) and Ki67 (red). Percentages of GFP+Ki67+ cells of total GFP+ cells per field were graphed (bottom panel). (D) Confocal images of lung section at 14 dpi with nuclei labeled by DAPI (blue) and antibodies against GFP (green) and T1a (red). Asterisks indicate regions double-positive for GFP and T1a. (E) Lung cells were dissociated and flow cytometry was performed by gating on GFP+T1a+. The numbers in the top left gates represent all GFP+ cells of total live CD45– cells. The numbers in the top right gates represent GFP+T1a+ cells of total T1a+ cells. The numbers in the bottom right gates represent all T1a+ cells of total live CD45– cells. (F) Quantification of GFP+T1a+ cells as the percentage of GFP+T1a+ of total T1a+ cells by flow cytometry. n = 4–5 per group (B and C); n = 4–8 per group (F). **P < 0.01; ***P < 0.001; ****P < 0.0001, 2-way ANOVA. Scale bars: 25 μm (B and C); 20 μm (D).
Figure 5
Figure 5. Inflammatory responses in Yap/Taz mutant lungs.
(A) GFP+ AECIIs at 7 dpi were sorted by FACS and analyzed by qRT-PCR (n = 4 per group). (B) BALF was analyzed for IL-1b by ELISA assay (n = 4–6 per group). (C) Cytokine assay showed protein levels of IL-1b and CXCL9 in mouse lung lysates (n = 1 per group). (D) Flow cytometry of dissociated lung cells was performed by gating on CD3+CD45+ cells, and quantification of total number of CD3+CD45+ cells in the lung was graphed (n = 3–10 per group). (E) Confocal images of lung sections at 14 dpi with nuclei labeled by DAPI (blue) and antibodies to GFP (green) and CD3 (red). Scale bars: 20 μm. (F) Quantification of the number of CD3+ cells as the ratio of CD3+ cell number versus GFP+ area per field using ImageJ (n = 3–5 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Student’s t test (A) and 2-way ANOVA (B, D, F).
Figure 6
Figure 6. Regulation of Yap/Taz on IκBa expression and NF-κB transcriptional activity in AECIIs.
(A) Schematic of IκBa genomic locus showing potential Tead binding sites (TBM) in the 10 kb upstream of the IκBa transcription start site. (B) Chromatin from MLE-15 cells was immunoprecipitated with either Tead or Yap antibody, and qRT-PCR results were graphed (n = 3). (C) MLE-15 cells were transfected with pGL3 vector containing a IκBa-TBM1-3 or a IκBa-TBM1-3 mutation, in which TBM1 was mutated, along with the expression plasmid encoding either murine Tead2 or Yap or Taz. Twenty-four hours after transfection, cells were processed for luciferase activity measurement (n = 3–6 per group). (D) Adult WT mouse AECIIs were purified, cultured, and infected with either Yap shRNA lentivirus or scramble shRNA lentivirus or were given no treatment. Forty-eight hours after lentiviral infection, cells were processed for NF-κB transcription activity measurement. (E) AECIIs were purified from mouse lungs at 0 dpi and 7 dpi, and their NF-κB transcriptional activity was graphed. (F) MLE-15 cells were infected with either Yap shRNA lentivirus or scramble shRNA lentivirus or were given no treatment. Forty-eight hours after lentiviral infection, cells were transfected with NF-κB luciferase vector containing NF-κB response elements, along with an expression plasmid encoding either murine Yap or Taz. Twenty-four hours after transfection, cells were processed for luciferase activity measurement (n = 3). (G) Schematic model of interaction of Yap/Taz and IκBa/NF-κB in AECIIs. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA (B, C, D, F) and 2-way ANOVA (E).
Figure 7
Figure 7. Inflammatory resolution and alveolar epithelial recovery in Yap/Taz mutant lungs with AAV6-IκBa treatment.
(A) Schematic of experimental design for B–K. (B) BALF was analyzed for IL-1b by ELISA assay (n = 4–5 per group). (C) Cytokine assay showed protein levels of IL-1b and CXCL9 in mouse lung lysates (n = 1 per group). (D) Flow cytometry of dissociated lung cells was performed by gating on CD3+CD45+ cells, and quantification of total number of CD3+CD45+ cells in the lung at 14 dpi were graphed (n = 5–6 per group). (E) Immunostaining on lung sections at 14 dpi with nuclei labeled by DAPI (blue) and antibody to T1a (red). White dashed lines indicate T1a– region in the lung. (F) Flow cytometry of dissociated lung cells was performed by gating on T1a+CD45– cells. (G) Quantification of the percentage of T1a+CD45– cells of total CD45– cells in the lung at 14 dpi (n = 4–5 per group). (H) Quantification of total protein in BALF at 14 dpi (n = 5–6 per group). (I) Lung sections at 14 dpi were stained with Alcian blue and nuclear fast red. (J) Quantification of lung fibrotic regions at 14 dpi using Ashcroft scoring method (n = 4–6 per group). (K) Quantification of collagen level on lung tissue lysates at 14 dpi using hydroxyproline assay (n = 4 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA. Scale bars: 50 μm.
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