PURPOSE: Idiopathic pulmonary fibrosis (IPF) is the most common and most aggressive form of idiopathic interstitial pneumonia. Transforming growth factor beta (TGF-β) has been shown to be a particularly important mediator of fibroblast proliferation and extracellular matrix (ECM) production in IPF lungs. Interferon gamma-1b (IFN-γ 1b) inhibits TGF-β-induced collagen synthesis by up-regulating SMAD7 expression and inducing formation of an inhibitory RFX5 complex on the collagen promoter region. We recently discovered that pirfenidone exerts antifibrotic effects by inhibiting p38 MAPK, an essential intracellular mediator of TGF-β-induced collagen synthesis. Given their distinctive modes of action against TGF-β-induced collagen expression, we wanted to determine whether the concomitant administration of IFN-γ 1b and pirfenidone would result in complementary or antagonistic effects against TGF-β-induced gene induction in human lung fibroblasts (HFL-1).
METHODS: HFL-1 cells were pre-treated with pirfenidone (185 mg/L) and IFN-γ 1b (300 pg/mL) for 1 hour, then TGF-β was added and cells were incubated for 24 hours in serum-containing complete FK12 growth medium. Total RNA isolation, gene chip hybridization, Western blot and ELISA were performed according to manufacturer protocols.
RESULTS: Analyses of oligonucleotide micro arrays identified 110 genes that were up-regulated between two and sixty-five fold (p=0.001) at 24 hours after TGF-β addition. Both pirfenidone and IFN-γ 1b inhibited TGF-β-induced gene induction to varying degrees, while the combination of these agents resulted in additive suppression of all TGF-β-induced genes. We used Western blot and ELISA to measure protein expression levels for a panel of profibrotic molecules. Results confirmed that the additive effect of IFN-γ 1b and pirfenidone against TGF-β-induced gene induction was maintained at the level of protein expression.
CONCLUSION: The co-administration of IFN-γ 1b and pirfenidone does not appear to antagonize the individual activity of either compound against TGF-β-induced gene induction <i>in vitro</i> and may result in additive suppression of collagen synthesis and ECM production.
CLINICAL IMPLICATIONS: Pending positive results of clinical trials evaluating these drugs individually, further work to elucidate the effect of both drugs in combination may be warranted.
DISCLOSURE: Osman Ozes, University grant monies No; Grant monies (from sources other than industry) No; Grant monies (from industry related sources) T. Takuma and M. Taylor: Grant from InterMune; Shareholder O. Ozes, J. Derrick, S. Stevens, L. Blatt: InterMune shareholders; Employee O. Ozes, J. Derrick, S. Stevens, L. Blatt: InterMune employees; Fiduciary position (of any organization, association, society, etc, other than ACCP No; Consultant fee, speaker bureau, advisory committee, etc. No; Other No; Product/procedure/technique that is considered research and is NOT yet approved for any purpose, All.