The inflammatory eye disease Graves’ orbitopathy (GO) is the main complication of autoimmune Graves’ disease. In previous studies we have shown that hypoxia plays an important role for progression of GO. Hypoxia can maintain inflammation by attracting inflammatory cells such as macrophages (MQ). Herein, we investigated the interaction of MQ and orbital fibroblasts (OF) in context of inflammation and hypoxia. We detected elevated levels of the hypoxia marker HIF-1α, the MQ marker CD68, and inflammatory cytokines TNFα, CCL2, CCL5, and CCL20 in GO biopsies. Hypoxia stimulated GO tissues to release TNFα, CCL2, and CCL20 as measured by multiplex enzyme-linked immunosorbent assay (ELISA). Further, TNFα and hypoxia stimulated the expression of HIF-1α, CCL2, CCL5, and CCL20 in OF derived from GO tissues. Immunofluorescence confirmed that TNFα-positive MQ were present in the GO tissues. Thus, interaction of M1-MQ with OF under hypoxia also induced HIF-1α, CCL2, and CCL20 in OF. Inflammatory inhibitors etanercept or dexamethasone prevented the induction of HIF-1α and release of CCL2 and CCL20. Moreover, co-culture of M1-MQ/OF under hypoxia enhanced adipogenic differentiation and adiponectin secretion. Dexamethasone and HIF-1α inhibitor PX-478 reduced this effect. Our findings indicate that GO fat tissues are characterized by an inflammatory and hypoxic milieu where TNFα-positive MQ are present. Hypoxia and interaction of M1-MQ with OF led to enhanced secretion of chemokines, elevated hypoxic signaling, and adipogenesis. In consequence, M1-MQ/OF interaction results in constant inflammation and tissue remodeling. A combination of anti-inflammatory treatment and HIF-1α reduction could be an effective treatment option.

Background: Graves’ disease (GD) is an autoimmune condition that can extend beyond the thyroid, leading to thyroid eye disease (TED), a disorder marked by orbital inflammation and tissue remodeling. Methods: We explored the therapeutic potential of maraviroc, a CCR5 antagonist, in a mouse model of TED triggered by immunization with the human TSH receptor (hTSHR) A-subunit. Mice received pTriEx1.1neo-hTSHR A-subunit plasmid immunizations, and a subset were treated with maraviroc via drinking water. We assessed thyroid function, orbital tissue changes, immune cell infiltration, and lipid metabolism through serological testing, histology, immunohistochemistry, and untargeted lipidomics. Results: Maraviroc did not significantly affect anti-TSHR antibody production nor the degree of hyperthyroidism, though it modestly improved thyroid histopathology. Notably, it reduced key signs of orbital disease, including brown adipose tissue expansion, CCL5-positive immune cell infiltration, CD4 ⁺ T-cell infiltration and the presence of F4/80 ⁺ macrophages. Lipidomic profiling revealed distinct metabolic changes in treated mice, with reduced triacylglycerols and elevated carnitines, indicative of enhanced fatty acid utilization. Composite Z-score analysis reinforced maraviroc’s beneficial effects on orbital inflammation and remodeling. Conclusion: Maraviroc shows promise as a targeted therapy for TED in the context of GD, offering anti-inflammatory and anti-adipogenic benefits while sparing thyroid autoimmunity. These preclinical findings support further clinical investigation into its role in managing TED.

There is an unmet need to identify biomarkers of active thyroid eye disease (TED). scRNAseq revealed that orbital fibroblasts from orbital decompressions in people with TED express high levels of thyroid hormone receptors, growth factor receptors, including insulin-like growth factor 1 receptor (IGF1R), and extracellular matrix proteins including SPARC (osteonectin), whereas orbital fat endothelial cells expressed thyroid peroxidase (TPO). SPARC was significantly raised in the serum of people with thyroid disease compared to healthy controls. Furthermore, those with moderate, severe and sight threatening TED had higher SPARC levels than those with thyroid disease but free of TED or mild TED. Free-triiodothyronine (FT3) levels were positively correlated with SPARC in moderate-sight threatening TED. SPARC and IGF1 were positively correlated across people with thyroid disease alone, as well as TED. Thyroid stimulating hormone (TSH) levels were negatively correlated with SPARC in moderate-sight threatening TED. When participants were followed longitudinally, SPARC decreased after the active phase of TED. At the protein level, immunohistochemistry indicated that SPARC was heterogeneously expressed by fibroblasts in both control and TED orbital fat. SPARC is a key mediator of fibrosis and deposition of extracellular matrix and the correlation of SPARC serum levels to TED status and FT3 make it a promising biomarker of active TED.

Background: Thyroid eye disease (TED) is characterized by orbital inflammation, fibroblast activation, and pathological tissue remodeling. While autoimmunity to the thyrotropin receptor (TSHR) initiates disease, the contribution of proinflammatory cytokines, such as tumor necrosis factor (TNFα), to fibroblast differentiation and extracellular matrix (ECM) remodeling remains incompletely understood. Methods: A mouse model of TED was generated by immunization with a plasmid encoding the human TSHR A-subunit. Orbital tissues were analyzed for immune cell infiltration and cytokine expression. Primary murine orbital fibroblasts (mOFs) from TSHR- and control β-Gal-immunized mice were treated with TNFα or IFNγ. The expression of cytokines, chemokines, adiponectin, TGFβ, hyaluronic acid (HA), and hyaluronan synthase 2 (HAS2) was assessed by ELISA, qPCR, and western blotting. Results: TSHR-immunized mice displayed marked orbital macrophage infiltration accompanied by elevated TNFα levels. TNFα induced a strong proinflammatory response in mOFs, with robust IL-6 and IL-8 secretion and an IL-6-dominant profile in TSHR-derived cells. TNFα also upregulated CCL2, CCL20, and CXCL10, supporting enhanced immune cell recruitment. Moreover, TNFα reduced adiponectin while increasing TGFβ in TSHR mOFs, indicating activation of profibrotic pathways. TNFα significantly increased HA production and HAS2 expression, particularly in TSHR-derived fibroblasts, demonstrating enhanced ECM synthesis under autoimmune conditions. Conclusion: These findings identify TNFα as a central regulator linking inflammation to adipogenic suppression, profibrotic signaling, and ECM remodeling in a TSHR-immunized mouse model of TED. Enhanced HA/HAS2 induction underscores a disease-specific sensitivity of orbital fibroblasts to TNFα. Targeting TNFα or its downstream IL-6/TGFβ–ECM axis may offer a promising therapeutic strategy to limit pathological tissue remodeling in TED.