Cutting Edge: Retrobulbar Inflammation, Adipogenesis, and Acute Orbital Congestion in a Preclinical Female Mouse Model of Graves’ Orbitopathy Induced by Thyrotropin Receptor Plasmid-in Vivo Electroporation

<p>Graves' orbitopathy (GO) is a complication in Graves' disease (GD) but mechanistic insights into pathogenesis remain unresolved, hampered by lack of animal model. The TSH receptor (TSHR) and perhaps IGF-1 receptor (IGF-1R) are considered relevant antigens. We show that genetic immunization of human TSHR (hTSHR) A-subunit plasmid leads to extensive remodeling of orbital tissue, recapitulating GO. Female BALB/c mice immunized with hTSHR A-subunit or control plasmids by in vivo muscle electroporation were evaluated for orbital remodeling by histopathology and magnetic resonance imaging (MRI). Antibodies to TSHR and IGF-1R were present in animals challenged with hTSHR A-subunit plasmid, with predominantly TSH blocking antibodies and were profoundly hypothyroid. Orbital pathology was characterized by interstitial inflammation of extraocular muscles with CD3+ T cells, F4/80+ macrophages, and mast cells, accompanied by glycosaminoglycan deposition with resultant separation of individual muscle fibers. Some animals showed heterogeneity in orbital pathology with 1) large infiltrate surrounding the optic nerve or 2) extensive adipogenesis with expansion of retrobulbar adipose tissue. A striking finding that underpins the new model were the in vivo MRI scans of mouse orbital region that provided clear and quantifiable evidence of orbital muscle hypertrophy with protrusion (proptosis) of the eye. Additionally, eyelid manifestations of chemosis, including dilated and congested orbital blood vessels, were visually apparent. Immunization with control plasmids failed to show any orbital pathology. Overall, these findings support TSHR as the pathogenic antigen in GO. Development of a new preclinical model will facilitate molecular investigations on GO and evaluation of new therapeutic interventions.</p

Genetic immunization with mouse thyrotropin hormone receptor plasmid breaks self-tolerance for a murine model of autoimmune thyroid disease and Graves' orbitopathy

Experimental models of Graves' hyperthyroid disease accompanied by Graves' orbitopathy (GO) can be efficiently induced in susceptible inbred strains of mice by immunization by electroporation of heterologous human TSH receptor (TSHR) A-subunit plasmid. In this study, we report on the development of a bona fide murine model of autoimmune Graves' disease induced with homologous mouse TSHR A-subunit plasmid. Autoimmune thyroid disease in the self-antigen model was accompanied by GO and characterized by histopathology of hyperplastic glands with large thyroid follicular cells. Examination of orbital tissues showed significant inflammation in extraocular muscle with an accumulation of T cells and macrophages together with the substantial deposition of adipose tissue. Notably, increased levels of brown adipose tissue were present in the orbital tissue of animals undergoing experimental GO. Further analysis of inflammatory loci by ¹⁹F-magnetic resonance imaging showed inflammation to be confined to orbital muscle and optic nerve, but orbital fat showed no difference in inflammatory signs in comparison to control ß-Gal immunized animals. Pathogenic antibodies induced to mouse TSHR were specific for the self-antigen, with minimal cross-reactivity to human TSHR. Moreover, compared to other self-antigen models of murine Graves' disease induced in TSHR knock out mice, the repertoire of autoantibodies to mouse TSHR generated following the breakdown of thymic self-tolerance is different to those that arise when tolerance is not immunologically breached as in the knock out models. Overall, we show mouse TSHR A-subunit plasmid immunization by electroporation overcomes tolerance to self-antigen to provide a faithful model of Graves' disease and GO. This article is protected by copyright. All rights reserved