Endocrine Abstracts

Introduction: During the course of thyroid eye disease (TED) immune cells infiltrate the orbital connective tissue leading to overstimulation of orbital fibroblasts (OFs). In the persistent inflammatory environment, OFs differentiate into myofibroblasts or adipocytes, simultaneously creating two phenotypes with different morphology and function. Activated OFs secrete pro-inflammatory cytokines and growth factors and contribute to the prolonged inflammation, among other things by producing chemotactic proteins. Chemerin is a potent chemoattractant that triggers chemotaxis of macrophages and natural killer cells, promotes proliferation and it stimulates adipogenesis; its expression by OFs has not been studied yet. Our aim was to study the production of chemerin by OFs and during their differentiation into adipocytes and pro-fibrotic myofibroblasts.

Methods: We used primary cultures of OFs established from orbital connective tissue samples of patients with TED (n = 5). Myofibroblast differentiation was induced using 5 ng/ml TGF-β1 for 72 hours and verified by measuring α-smooth muscle actin (α-SMA) expression using RT-PCR. Adipogenic differentiation was induced and maintained for 12 days by adipogenic medium and lipid accumulation was measured by Oil Red O (ORO) staining. Supernatants were collected, then processed with Human Chemerin ELISA (R&D Systems, DY2324) commercial kit to detect chemerin production.

Results: We found that OFs originated from TED patients produced chemerin. Chemerin production was increased during TGF-β1-induced myofibroblast differentiation after 72 hours (mean fold increase ± SD: 3.4 ± 1.5, P = 0.019). On the other hand, the concentration of chemerin decreased in the cell culture supernatant during adipogenesis (concentration ± SD: 1.7 ± 0.8, 1.3 ± 0.6, 0.7 ± 0.6, 0.8 ± 0.5 ng/ml at days 0, 4, 8 and 12, respectively; P < 0.0001). Myofibroblast and adipocyte differentiations were confirmed by increased α-SMA expression and lipid accumulation, respectively.

Conclusions: In this study we have shown that orbital fibroblasts secrete chemerin and chemerin production increases after myofibroblast differentiation. These findings suggest that myofibroblasts may promote macrophage infiltration into the orbit which, according to the phenotype of macrophages, can perpetuate the inflammation or induce additional myofibroblast differentiation by secreting TGF-β. Although chemerin is known as an adipokine, adipogenesis in OFs has led to decreased chemerin expression. Further studies are needed to clarify the role of chemerin in the pathogenesis of TED.