Cigarette Smoking Drives Thyroid Eye Disease Progression via RAGE Signaling Activation

Background: Thyroid eye disease (TED) is a sight-threatening autoimmune disease with cigarette smoking as one of the key risk factors. Cigarette smoking affects both the severity of TED and the patient's response to medication. However, the underlying pathogenic mechanisms of smoking in TED remain unclear. Methods: Orbital fibroblasts (OFs) were extracted from patients with TED and non-TED controls, and treated with cigarette smoking extract (CSE). Luminex assays and Western blots were employed to examine inflammatory status and pathological phenotypes of OFs. A specific reactive oxygen species (ROS) probe was used to evaluate oxidative stress levels. RNA-sequencing of CSE-treated OFs was used to analyze differentially expressed genes. Immunofluorescence and RNA-sequencing were used to examine the expression of receptor for advanced glycation end products (RAGE) signaling molecules in patients. Small interfering RNA sequences and a RAGE-specific inhibitor were employed to investigate the effects of RAGE blockade on cigarette smoking-related pathological phenotypes. To validate our findings in vivo, we generated an adenovirus-induced TED mouse model with exposure to cigarette smoke. Results: Exposure to CSE resulted in an inflammatory phenotype of OFs together with higher levels of oxidative stress. OFs exposed to CSE presented susceptibility to transforming growth factor-β-induced myofibroblast differentiation, and 15-D-PGJ₂-induced adipocyte differentiation, indicating pro-fibrotic and pro-adipogenic phenotypes. RNA-sequencing of CSE-treated OFs revealed upregulation of RAGE signaling molecules. TED patients with smoking history also exhibited higher levels of RAGE signaling, both in the orbit and peripheral blood, compared with non-smoking patients. Enhancement of inflammatory status was associated with activation of the ROS-nuclear factor-kappa B pathway downstream of RAGE. RAGE gene interference or administration of RAGE inhibitor effectively mitigated cigarette smoking-related pathological changes in OFs. Disrupting RAGE signaling in TED mice efficiently ameliorated smoking-induced disease progression in vivo. Conclusions: Cigarette smoking-relevant TED progression was linked with RAGE signaling activation, leading to the exacerbation of orbital inflammation and tissue-remodeling, including fibrosis and adipogenesis. Our findings demonstrate that cigarette smoke exposure affects the biological characteristics of TED-derived OFs and supports RAGE as a promising therapeutic target for the management of patients with TED and smoking habits.