The Pathogenic Role of Smooth Muscle Cell-Derived Wnt5a in a Murine Model of Lung Fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by extensive fibrosis of the lung tissue. Wnt5a expression was observed to be upregulated in IPF and suggested to be involved in the progression of the disease. Interestingly, smooth muscle cells (SMC) are a major source of Wnt5a in IPF patients. However, no study has been conducted until now to investigate the precise role of smooth muscle-derived Wnt5a in IPF. Here, we used the bleomycin-induced lung fibrosis model in a conditional gene-deficient mouse, where the Wnt5a gene was excised from SMC. We show here that the excision of the Wnt5a gene in SMC led to significantly improved health conditions with minimized weight loss and improved lung function. This improvement was based on a significantly lower deposition of collagen in the lung with a reduced number of fibrotic foci in lung parenchyma. Furthermore, the bleomycin-induced cellular infiltration into the airways was not altered in the gene-deficient mice compared with wild-type mice. Thus, we demonstrate that the Wnt5a expression of SMC of the airways leads to aggravated fibrosis of the lung with poor clinical conditions. This aggravation was not an influence in the bleomycin-induced inflammatory processes but on the development of fibrotic foci in lung parenchyma and the deposition of collagen.

Keywords: Wingless-Type MMTV integration site family, member 5A; airway smooth muscle; bleomycin; fibrotic foci; pulmonary fibrosis.

Figure 1

Clinical symptoms of SMC-derived Wnt5a knockout (KO) and wild-type (WT) mice after the inoculation of the saline solution or bleomycin (Bleo). (A) Mice weight variation through the experiment is represented in percentage values, where the day of bleomycin (Bleo) or saline solution (day 1) administration was chosen as the reference (100%). (B) Mice were supervised every day and a score was given according to the different clinical signs, such as weight loss, behavioral change and altered respiratory frequency. Mice were narcotized on the last day of the experiment and subjected to lung function measurements through the flexiVent system; where (C) the pressure–volume curve was performed in a pressure–driven way, and (D) the inspiratory capacity volume was obtained automatically from the flexiWare software from the pressure-volume curve. All data are shown as mean ± SEM per group, */# p < 0.05; **/## p < 0.01 by two-way ANOVA with the Bonferroni post-test (C) and Mann–Whitney test (D). Statistics between KO and WT mice in Figure C are represented with an #. n = 6 (KO + NaCl), n = 10 (KO + Bleo), n = 6 (WT + NaCl) and n = 9 (WT + Bleo). The experiment was repeated twice with similar results.

Figure 2

Analysis of collagen expression and deposition in bleomycin-induced lung fibrosis in KO and WT mice. (A) Images were recorded of the whole lung sections (scale: 1000 µm) and of proximal bronchi (scale: 500 µm) stained with Sirius red, where (B) the collagen area in the parenchyma was extrapolated as a percentage and (C) the thickness of the collagen layer in the proximal peribronchial was measured in µm. Additionally, (D) the collagen content was measured in bronchoalveolar lavage fluid (BALF) samples with a Sircol^TM assay. The experiment was repeated twice with similar results. All data are shown as mean ± SEM per group, * p < 0.05; ** p < 0.01; *** p < 0.001 by Mann–Whitney test.

Figure 3

Histological analysis of αSMA-positive cells. (A) Expression of reporter fluorescence protein tdTomato (red) in αSMA positive cells was analyzed in fixed frozen sections of lung with 4% PFA and covered with medium DAPI (blue); images were recorded (scale: 200 µm). (B) The peribronchial muscle layer of proximal bronchi was stained with antibody against αSMA and a secondary antibody coupled with AF488 in paraffin-embedded lung sections (scale: 100 µm). (C) Representation of an αSMA positive cell (green arrow) with typical spindle-shape and dendritic morphology of myofibroblasts (scale: 100 µm). Both immunostainings were evaluated with the help of ImageJ software, (D) rate of tdTomato expressing cells were evaluated by recording at least eight images of the parenchyma, where airways and blood vessels were avoided. The total area of cells expressing tdTomato (red) as a percentage was obtained and calculated referring to the total area of cells stained with DAPI (blue) as a percentage: Rate = (% Area expressing tdTomato × 100)/% Area expressing DAPI. (E) The thickness of the peribronchial muscle layer was obtained by measuring five random pictures of proximal bronchi per mouse. All data are shown as mean ± SEM per group, ** p < 0.01; *** p < 0.001 by Mann–Whitney test.

Figure 4

Analysis of inflammatory response upon bleomycin-induced lung fibrosis in KO and WT mice. (A) Total number of macrophages, lymphocytes and neutrophils in BALF samples from KO treated with saline solution (column filled with small squares) and bleomycin (bigger squares) and WT mice with saline (horizontal lines) or bleomycin inoculation (vertical line). The same lung lobe from each mouse was removed and homogenized in PBS containing proteinase inhibitors, (B) IL-6, (C) TNFα and (D) TGF-β were measured in the lung homogenate with ELISA and are represented in pg/mL in each graph. (E) Due to the high increase of IL-6 in WT compared to KO mice, IL-6 was correlated with compliance of the respiratory system (C_rs) obtained from the lung function (Figure S2C). The experiment was repeated twice with similar results. All data are shown as mean ± SEM per group, * p < 0.05; ** p < 0.01; *** p < 0.001 by Mann–Whitney test.

Figure 5

Experimental plan and confirmation of Wnt5a excision. The experiment was performed with four distinguishable groups: 1. Knockout (KO) + NaCl—Control Wnt5a KO mice that were treated with tamoxifen and saline solution i.n.; 2. KO + Bleo—Wnt5a KO mice that were given tamoxifen and lung fibrosis was induced with bleomycin; 3. WT + NaCl—Control reference mice (iSma-Cre) that were given only tamoxifen and saline solution i.n.; 4. WT + Bleo—Reference mice that were given tamoxifen and lung fibrosis was induced with bleomycin. Mice that only express Cre enzyme and tdTomato upon tamoxifen injection (iSma-Cre mice) were used as WT, due to no conditional KO of any gene. (A) All mice were treated with tamoxifen, lung fibrosis was induced with bleomycin in KO and WT mice on day 0, and control KO and WT mice treated with saline solution on day 1. Lung function measurement and mouse preparation was done exactly three weeks after the i.n. inoculation of bleomycin and saline solution. (B) Ear tissue from WT and KO mice was taken before and after tamoxifen administration to demonstrate the KO of the Wnt5a gene (268 bp) in KO mice and wild-type phenotype in WT mice (223 bp) after tamoxifen injection. The KO mice still had many cells where the gene was only floxed (351 bp), due to the higher number of αSMA negative cells.