Pirfenidone attenuates the interleukin-1β-induced hyaluronic acid increase in orbital fibroblasts of thyroid-associated ophthalmopathy

Abstract

Thyroid-associated ophthalmopathy (TAO) is an autoimmune component of Graves’ disease characterized by intense inflammation in the setting of volume expansion and tissue remodeling, leading to organ dysfunction. At the heart of this, tissue remodeling occurred by the disordered accumulation of glycosaminoglycan; hyaluronic acid (HA), which results in tissue edema due to its strong hydrophilic property. Because no reliable, specific and safe medical therapeutic agents are available for TAO, the development of specific therapies with minimal side effects is essential. Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) is a novel agent that has shown its anti-inflammatory and anti-fibrotic effects in animal models and clinical trials.

We investigated an effect of pirfenidone on interleukin (IL)-1β-induced HA expression in an in vitro model of TAO. The effect of pirfenidone on IL-1β-induced HA expression was assessed in primary cultured orbital fibroblasts of TAO patients. The level of HA in IL-1β-treated cells with or without pirfenidone was measured using an enzyme-linked immunosorbent assay (ELISA). The effect of pirfenidone on IL-1β-induced hyaluronic acid synthase (HAS) expression was evaluated by reverse transcription-polymerase

chain reaction (RT-PCR), and verified by Western blot. We then examined the role of mitogen-activated protein kinase (MAPK)s on IL-1β-induced HAS expression by RT-PCR, using specific inhibitors to p38 MAPK (p38), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The level of phosphorylation of MAPKs in IL-1β-treated cells with or without pirfenidone was measured by immunoblot analysis. In addition, the effect of pirfenidone on HA production was compared with that of dexamethasone by ELISA.

Pirfenidone strongly attenuated the IL-1β-induced HA release with a dose -dependent manner. IL-1β-induced HAS mRNA and protein expression decreased significantly by co-treatment with pirfenidone. In the signaling pathway of IL-1β, HAS transcription was mediated by p38 and ERK dependent pathways, and the phosphorylations of them were nicely attenuated by co-treatments with pirfenidone. In our system, JNK pathways was not related with IL-1β-induced HAS transcription. Finally, pirfenidone was more effective than dexamethasone in inhibiting IL-1β-induced HA increases.

In summary, pirfenidone attenuates the IL-1β-induced HA production in orbital fibroblasts from patients with TAO, which is related to suppression of the MAPK-mediated HAS expression. These results support the potential use of pirfenidone in treatment of TAO.