Polymorphisms in the IL-1 Gene Family and Lung Disease Severity in Patients with Cystic Fibrosis

Effective treatments for idiopathic pulmonary fibrosis (IPF) are not yet available. Current concepts indicate that multi-agent synergistic drug regimens will be needed to control this lethal condition. The available animal models for lung fibrosis are laborious, variably reproducible, and do not faithfully represent the histopathologic features of IPF. To address this, we developed a new in vitro assay to screen potential drugs for synergistic activity against lung fibrosis. Our group was among the first to implicate transforming growth factor-b1 (TGF-b1) as a principal growth factor in mediating IPF. Additional data indicate roles for platelet-derived growth factor (PDGF) and tumor necrosis factor-a (TNF-a) in the fibrotic process. Accordingly, we cultured IMR90 human lung fibroblasts in the presence of cytokines, such as TGF-b1 (5 ng/ml). The generation of extracellular matrix fibronectin and collagen 1 was quantified by real-time PCR using the iCycler system (Bio-Rad, Hercules, CA), and confirmed with enzyme-linked immunosorbent assay. With this approach we undertook studies of oral agents with activity against cytokinedriven fibrosis. Addition of pentoxifylline (an anti–TNF-a agent) and pirfenidone (an agent with activity against TGF-b1 and PDGF), each individually significantly reduced collagen and fibronectin expression. However, together the combination yielded nearly complete suppression of cytokine-stimulated matrix expression. This synergistic regimen also displayed no abnormal toxicity on the cultured Institute for Medical Research (IMR) lung fibroblasts. Currently, we are testing another drug combination that includes the PPAR-g agonist, Rosiglitazone, along with pentoxifylline, and have shown similar synergistic effects to that of pirfenidone and pentoxifylline. Finally, for both drug combinations Western blotting with a-smooth muscle actin was conducted to determine if these two drug combinations prevented fibroblast to myofibroblast differentiation phenotype. Results from these experiments showed no effect on a-smooth muscle protein levels, suggesting that these drug combinations are acting downstream and independently of SMADs. In conclusion, we believe that this rapid screening approach will be useful to efficiently identify drug combinations with greater activity against fibrosis. Selected combinations can be subsequently tested in available animal models, and ultimately recommended for clinical trials in humans.