Bleomycin induces molecular changes directly relevant to idiopathic pulmonary fibrosis: a model for "active" disease

Abstract

The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.

Figure 1. Bleomycin induces inflammatory and fibrotic changes in the lungs of mice.

The numbers of BALF inflammatory cells increased after either a single (A) or repetitive (D bleomycin (black bars) instillation compared to the saline treated controls (white bars). Both a single (B) and repetitive (E) instillation of bleomycin led to increased fibrotic tissue in the lung. Changes in work of inflation (WoI), a measure of lung mechanics, were minimal in both the single (C) and repetitive (F) systems. Data are expressed as mean ± SEM of n = 7–8 mice. Significance (relative to the time-matched control at each time point) was determined using a Student’s t-test and is denoted as follows: *p<0.05; **p<0.01; and ***p<0.001.

Figure 2. Bleomycin induces inflammation, fibrosis, and epithelial remodeling.

Representative images are shown from saline treated controls (A), as well as 7 days (B), 21 days (C), and 35 days (D) after a single bleomycin instillation. Additionally, representative images are shown from saline treated controls (E), as well as 2 months (F), 4 months (G), and 6 months (H) after the start of the repetitive bleomycin instillations. Areas of alveolar (black arrows) and interstitial (green arrows) fibrosis were clearly present in both models as were areas of bronchoalveolar hyperplasia (green arrow heads). There were also areas of partially collapsed (D) and thickened alveoli (G) that were often lined by cuboidal bronchoalveolar epithelial cells (black arrow heads) in both systems. At the latest time point examined (H), there were also partially collapsed septa, macrophages, and multinucleate giant cells observed in the alveolar space (yellow arrows). Images were captured at 200X magnification of H&E stained lung tissue sections.

Figure 3. Unsupervised hierarchical clustering of genes differentially expressed between bleomycin and saline treated mice across time points.

A union of 730 genes differentially expressed between bleomycin and saline treatments (fold-difference >2, false discovery rate (FDR) <0.05) were clustered across all mouse samples. Three distinct clusters of genes were revealed corresponding to phases of the bleomycin response, with most up-regulated genes being specific to the 7–14 days post-bleomycin treatment clusters. Inflammation phase (1–2 days) samples also clustered together and showed up-regulation of a subset of genes, while late fibrosis phase (21–35 days) samples also showed moderate up-regulation of a subset of genes.

Figure 4. Supervised hierarchical clustering of custom panels of genes across all mouse samples.

Clustering of genes was performed for a panel of matrix metalloproteases (MMPs) and lysyl oxidase-like (LOXL) enzymes (A), of regulators of TGFβ signaling (B), and genes encoding matrix proteins (C) where samples were ordered based on treatment and time point.

Figure 5. Heatmap of gene set enrichment of mouse bleomycin-induced signatures in clinical IPF datasets.

GSEA was performed for gene sets comprised of up-regulated genes in response to bleomycin at each time point from the mouse model. Enrichment of each gene set (denoted in rows) was determined against ranked lists of genes from clinical datasets comparing IPF vs. non-IPF conditions as well as IPF with acute exacerbation vs. stable IPF from two datasets (GSE2052, GSE10667, denoted in columns). Enrichment scores were plotted in a heatmap where gene sets enriched in IPF samples (nominal p<0.05, FDR <0.25) were denoted in red with intensity based on enrichment score (calculated in GSEA).

Figure 6. Analysis of leading edge gene subsets from GSEA of bleomycin-induced gene sets in IPF vs. non-IPF comparisons from two clinical cohorts (GSE2052, GSE10667).

Red-blue color bars denote genes ranked based on fold-change and FDR in IPF vs. non-IPF comparisons within datasets. Each vertical bar corresponds to bleomycin-induced genes from the 14 day time point, with bar height corresponding to running enrichment score calculated in GSEA. Green bars correspond to genes in the leading edge subset, or those genes contributing most to enrichment of the gene set in human samples. A union of the leading edge gene subsets from the 3 enrichment analyses of IPF vs. non-IPF clinical samples was subsequently compared against canonical pathways to determine enriched pathways of bleomycin genes most altered in clinical IPF. The table illustrates the top 5 canonical pathways enriched among the leading edge gene subsets.

Figure 7. The ALK-5 inhibitor, SB525334, attenuates bleomycin-induced lung fibrosis.

SB525334 was administered prophylactically (A) or therapeutically (B) at a dose of 60 mg/kg in chow beginning 1 day prior or 5 days after bleomycin instillation, respectively. SB525334 inhibited the lung fibrosis under both dosing conditions. Data are expressed as a percentage of the total collagen I stained area in the bleomycin vehicle control group ± SEM of n = 10–12 bleomycin treated mice in the prophylactic study and n = 6–9 in the therapeutic study. Saline treated control groups consisted of n = 5–7 mice. Significance (relative to the bleomycin vehicle control) was determined using a 2-tailed t-testand is denoted as **p<0.01 or ***p<0.001.