Time-course transcriptome analysis of a double challenge bleomycin-induced lung fibrosis rat model uncovers ECM homoeostasis-related translationally relevant genes

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is an irreversible disorder with a poor prognosis. The incomplete understanding of IPF pathogenesis and the lack of accurate animal models is limiting the development of effective treatments. Thus, the selection of clinically relevant animal models endowed with similarities with the human disease in terms of lung anatomy, cell biology, pathways involved and genetics is essential. The bleomycin (BLM) intratracheal murine model is the most commonly used preclinical assay to evaluate new potential therapies for IPF. Here, we present the findings derived from an integrated histomorphometric and transcriptomic analysis to investigate the development of lung fibrosis in a time-course study in a BLM rat model and to evaluate its translational value in relation to IPF.

Methods: Rats were intratracheally injected with a double dose of BLM (days 0-4) and sacrificed at days 7, 14, 21, 28 and 56. Histomorphometric analysis of lung fibrosis was performed on left lung sections. Transcriptome profiling by RNAseq was performed on the right lung lobes and results were compared with nine independent human gene-expression IPF studies.

Results: The histomorphometric and transcriptomic analyses provided a detailed overview in terms of temporal gene-expression regulation during the establishment and repair of the fibrotic lesions. Moreover, the transcriptomic analysis identified three clusters of differentially coregulated genes whose expression was modulated in a time-dependent manner in response to BLM. One of these clusters, centred on extracellular matrix (ECM)-related process, was significantly correlated with histological parameters and gene sets derived from human IPF studies.

Conclusions: The model of lung fibrosis presented in this study lends itself as a valuable tool for preclinical efficacy evaluation of new potential drug candidates. The main finding was the identification of a group of persistently dysregulated genes, mostly related to ECM homoeostasis, which are shared with human IPF.

Keywords: Interstitial Fibrosis.

Figure 1

BLM-induced lung fibrosis in the rat. (A) Workflow and timeline of the experiment. At each time point, at least six animals per group were used. (B) Representative Masson’s Trichrome-stained lung tissue sections at the starting point of the experiment (naïve animals, T0) and at 7, 14, 21, 28 and 56 days after the first BLM instillation. (C) Quantification of pulmonary fibrosis using the automated analysis (I) and Ashcroft Score evaluation (II) in BLM-treated and naïve animals. The mean values of % fibrosis and Ashcroft Score recorded in the BLM groups always significantly differed from the corresponding values measured in the SAL groups (p≤0.05). (D) Quantification of hydroxyproline level in lung homogenates obtained at the indicated time points from animals belonging to the SAL and BLM groups. The horizontal grey line represents the naïve (T0) level. The results were expressed as the mean±SEM; *p<0.05 BLM versus SAL. BLM, bleomycin; SAL, saline solution.

Figure 2

Transcriptomic profile of the rat lung in response to BLM treatment. (A) Graphical representation of sample distribution by PCA. (B) Bar graphs representing the number of upregulated and downregulated genes at each time point. (C) Heatmap of all genes deregulated in at least one time point. The hierarchical clustering of the columns was performed using a Euclidean distance metric. BLM, bleomycin; PCA, principal component analysis; SAL, saline solution.

Figure 3

Time-dependent transcription profiles following BLM administration. (A) Heatmap of the most informative modules (represented by their module eigengene, MEs). Each square represents the eigengene expression of a single biological condition (column) in a specific ME (rows). The left part of the heatmap represents comparisons between SAL or BLM at each time point with T0 (log2 FC (SAL/T0) and log2 FC (BLM/T0), respectively), while the right part shows comparisons between SAL and BLM (log2 FC (BLM/SAL)), as reported above the columns label. The eigengene expression was calculated as the ratio between the average eigengene expression of the biological replicates and the eigengene expression of the control group of interest (T0 or respective SAL). The last two columns on the right show the Pearson correlation value between each ME and the two histological parameters. Only the correlation values of ME8 and ME3 were statistically significant (log p-val −6). (B) Bar graphs of the most enriched terms identified by Metascape of the modules shown in A. Bar graphs represent the Log q-values and indicate the most significant terms for each group of similar terms. See online supplemental figure 1 and table 1 for detailed information on all WGCNA modules. BLM, bleomycin; SAL, saline solution; WGCNA, weighted correlation network analysis.

Figure 4

Association of gene signatures from time dependent BLM-induced rat lung with different human IPF samples detected by gene set enrichment analysis. The degree of enrichment of each analysed gene set (identified by the GEO accession number followed by a short description of the list) was determined against lists of preranked genes based on module membership (for the columns referred to the modules of the WGCNA on the left part of the image) or on log2 fold change (for the columns referred to the time points on the right part of the image). The NES is plotted in the heatmap with a scale form dark green (max negative NES) to dark orange (max positive NES). Grey: not significant NES (qval>0.05). BLM, bleomycin; IPF, idiopathic pulmonary fibrosis; WGCNA, weighted correlation network analysis.

Figure 5

Features of the most translationally relevant genes from module 8. Genes belonging to module 8, which are shared with the majority of human IPF studies (selected by GSEA leading edge analysis), are represented as a network to highlight their connections in terms of coexpression (light grey), colocalisation (dark grey) or genetic interaction (violet). Node size represents the correlation with the percentage of lung fibrosis; node colour represents the BLM induced (red) or repressed (blue) gene expression at T28. The table below gives a short gene description for each element of the figure. BLM, bleomycin; GSEA, gene set enrichment analysis; IPF, idiopathic pulmonary fibrosis.