Effects of phosphodiesterase 4 inhibition on bleomycin-induced pulmonary fibrosis in mice

Abstract

Background: Pulmonary fibrosis (PF) is a group of devastating and largely irreversible diseases. Phosphodiesterase (PDE) 4 is involved in the processes of remodeling and inflammation, which play key role in tissue fibrosis. The aim of the study was, therefore, to investigate the effect of PDE4 inhibition in experimental model of PF.

Methods: PF was induced in C57BL/6N mice by instillation of bleomycin. Pharmacological inhibition of PDE4 was achieved by using cilomilast, a selective PDE4 inhibitor. Changes in either lung inflammation or remodeling were evaluated at different stages of experimental PF. Lung inflammation was assessed by bronchoalveolar lavage fluid (BALF) differential cell count and reverse transcription quantitative polymerase chain reaction (RT-qPCR) for inflammatory cytokines. Changes in tissue remodeling were evaluated by pulmonary compliance measurement, quantified pathological examination, measurement of collagen deposition and RT-qPCR for late remodeling markers. Survival in all groups was analyzed as well.

Results: PDE4 inhibition significantly reduced the total number of alveolar inflammatory cells in BALF of mice with bleomycin-induced PF at early fibrosis stage (days 4 and 7). Number of macrophages and lymphocytes, but not neutrophils, was significantly reduced as well. Treatment decreased lung tumor necrosis factor (TNF)-alpha mRNA level and increased mRNA level of interleukin (IL)-6 but did not influence IL-1 beta. At later stage (days 14 and 24) cilomilast improved lung function, which was shown by increase in lung compliance. It also lowered fibrosis degree, as was shown by quantified pathological examination of Hematoxilin-Eosin stained lung sections. Cilomilast had no significant effect on the expression of late remodeling markers such as transforming growth factor (TGF)-beta1 and collagen type Ia1 (COL(I)alpha1). However, it tended to restore the level of lung collagen, assessed by SIRCOL assay and Masson's trichrome staining, and to improve the overall survival.

Conclusions: Selective PDE4 inhibition suppresses early inflammatory stage and attenuates the late stage of experimental pulmonary fibrosis.

Figure 1

PDE4 inhibition suppresses alveolar inflammation at early stage fibrosis. Total cell number, number of macrophages, lymphocytes and neutrophils in bronchoalveolar lavage fluid (BALF) of healthy controls treated with vehicle ("saline") and in mice suffering from fibrosis and treated either with vehicle ("bleo+ctrl") or cilomilast ("bleo+cilo") at days 4 and 7 after bleomycin instillation. Values are presented as means ± SEM, n = 6. * saline vs. bleo+ctrl (*p < 0.05, ***p < 0.001), † bleo+ctrl vs. bleo+cilo († p < 0.05, †† p < 0.01, ††† p < 0.001), # saline vs. bleo+cilo (# p < 0.05, ## p < 0.01).

Figure 2

Effect of PDE4 inhibition on lung inflammatory markers at early stage fibrosis. TNF-α, IL-6 and IL-1β mRNA levels in healthy controls treated with vehicle ("saline") and in mice suffering from fibrosis and treated either with vehicle ("bleo+ctrl") or cilomilast ("bleo+cilo") at days 4 and 7 after bleomycin administration. RT-qPCR data are normalized to β-actin expression and presented as ΔCt values ± SEM, n = 6. * saline vs. bleo+ctrl (***p < 0.001), † bleo+ctrl vs. bleo+cilo († p < 0.05, ††† p < 0.001), # saline vs. bleo+cilo (# p < 0.05, ### p < 0.001).

Figure 3

PDE4 inhibition improves lung function and attenuates tissue remodeling at late stage fibrosis. Lung compliance and fibrosis scores in healthy controls treated with vehicle ("saline") and in mice suffering from fibrosis and treated either with vehicle ("bleo+ctrl") or cilomilast ("bleo+cilo") at days 14 and 24 after bleomycin administration. Values are presented as means ± SEM, n = 9. * saline vs. bleo+ctrl (***p < 0.001), † bleo+ctrl vs. bleo+cilo († p < 0.05), # saline vs. bleo+cilo (### p < 0.001).

Figure 4

PDE4 inhibition attenuates tissue remodeling at late stage fibrosis. Representative images of lungs of healthy controls treated with vehicle (a, d) and of mice suffering from fibrosis and treated either with vehicle (b, e) or cilomilast (c, f) at days 14 (a, b, c) and 24 (d, e, f) after bleomycin administration. Hematoxilin-Eosin staining, magnification ×100.

Figure 5

Effect of PDE4 inhibition on lung collagen content at late stage fibrosis. SIRCOL assay for the lungs of healthy controls treated with vehicle ("saline") and in mice suffering from fibrosis and treated either with vehicle ("bleo+ctrl") or cilomilast ("bleo+cilo") at day 24 after bleomycin administration. Values are presented as means ± SEM, n = 4. * saline vs. bleo+ctrl (***p < 0.001), # saline vs. bleo+cilo (### p < 0.001).

Figure 6

Effect of PDE4 inhibition on survival. Healthy controls treated with vehicle ("saline") and mice suffering from fibrosis and treated either with vehicle ("bleo+ctrl") or cilomilast ("bleo+cilo") followed up for 24 days after bleomycin instillation. Kaplan-Meier curves, n = 9.