62 Chapter 3 RESULTS We observed COL14A1 expression in epithelial and stromal cells from control lung tissue in the HLCA dataset (Figure 1A), while expression of COL14A1 in the IPF atlas was mainly restricted to stromal cells (Figure 1B). Pseudobulk analysis performed on this dataset revealed that COL14A1 expression in bronchial epithelium, fibroblasts and myofibroblasts of IPF lungs was significantly higher compared to the same cells in non-IPF tissue (Figure 1C). Figure 1: Gene expression profile of COL14A1 in IPF and non-IPF lungs. A) COL14A1 expression in healthy lungs (data reconstructed from single-cell RNA-sequencing data obtained from Sikkema et al. [8]. B) COL14A1 expression in IPF and non-IPF lungs (reconstructed from single-cell RNA-sequencing data obtained from Adams et al. [9]), C) Comparison of COL14A1 counts in IPF and non-IPF lungs across relevant cell types: alveolar type 1 (AT1) and type 2 (AT2), bronchial epithelial cells (BE), fibroblasts (Fb), myofibroblasts (MyF) and smooth muscle cells (SMC). Data is represented as median ± 95% confidence interval. Applied statistical test: Mann-Whitney test. N=28 for non-IPF, n=32 for IPF. Immunohistochemical staining of the human lung tissue showed COL14A1 in control tissue, which was mainly localized in airways and parenchyma (Figure 2). Image analysis of whole lung tissue sections from IPF and non-IPF donors revealed relatively lower percentages of COL14A1 positive area in tissue from patients with IPF compared to those from both never and ex-smoker controls (Figure 3A). When focusing on specific regions, we also observed a relative lower percentage of positively-stained area in lung parenchyma of patients with IPF compared to both non-IPF control groups (Figure 3B), Lastly, the analysis of airways revealed a relative lower percentage of positive-stained area of COL14A1 in both airway wall (Figure 3C) and bronchial epithelium (Figure 3D) of IPF-derived lung tissue compared to non-IPF controls (both groups). In the whole tissue analysis and analyses of specific tissue compartments, there were no differences in relative positively-stained area between never-smoker and ex-smoker non-IPF tissues.
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