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

Abstract

Experimental models of Graves' hyperthyroid disease accompanied by Graves' orbitopathy (GO) can be induced efficiently 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 extra-ocular muscle with accumulation of T cells and macrophages together with 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 F-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 β-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 breached immunologically, as in the knock-out models. Overall, we show that mouse TSHR A-subunit plasmid immunization by electroporation overcomes tolerance to self-antigen to provide a faithful model of Graves' disease and GO.

Gut Microbiome in BALB/c and C57BL/6J Mice Undergoing Experimental Thyroid Autoimmunity Associate with Differences in Immunological Responses and Thyroid Function

“…The authors attributed the difference to high titres of blocking TSBAbs in the C57BL/6 mice [17]. However we are confident that our immunisation method, that is, plasmid immunisation with hTSHR A-subunit plasmid by electroporation, is superior to other modes of genetic delivery since in female BALB/c mice we were able to induce a strong immunological response and onset of both thyroid and orbital pathology, even resulting in breakdown of immune tolerance when using syngeneic antigen to induce disease [31]. The striking difference between the two strains of inbred mice immunised with hTSHR A-subunit plasmid by electroporation led us to examine the immunological profile in more detail.…”

Noninflammatory Diffuse Follicular Hypertrophy/Hyperplasia of Graves Disease: Morphometric Evaluation in an Experimental Mouse Model

“…Investigations involving animals have been approved by the institutional ethics animal committee of North Rhine Westphalia State Agency for Nature, Environment and Consumer Protection, Germany. At the age of 6–8 weeks, the mice were immunized with intramuscular injection and electroporation of 50 µg (1 mg/mL) plasmid into each biceps femoris muscle 4 times every 3 weeks [8, 9, 17]; 14 animals were immunized with pTRiEx1.1 human TSHR A-subunit plasmid and 17 animals with control ß-Gal plasmid (Ctrl). Six weeks after the end of immunization, the mice were sacrificed, and serum was collected and stored at –80°C.…”

“…The autoantigen that drives the immune response in GD is not the full-length TSHR, but the A-subunit, an ectodomain component that is shed after intramolecular cleavage of the receptor [16]. Genetic immunization of female inbred mice by electroporation with plasmid encoding human TSHR A-subunit cDNA, or indeed autologous mouse TSHR A-subunit, results in the generation of pathogenic cross-reactive antibodies that cause thyroid stimulation, thyroid enlargement with lymphocytic infiltration, elevated thyroxine levels, and in a subset of mice, ocular signs reminiscent of GO [12, 13, 17]. Additionally, the GD histopathological changes are heterogeneous and subtle in many cases, including a mixture of the initial hyperplastic reaction, along with the changes induced by the presurgical therapeutic intervention (e.g., Lugol) or antithyroid drug therapy.…”

Central Tolerance Mechanisms to TSHR in Graves’ Disease: Contributions to Understand the Genetic Association

“…Therefore, it is possible that split tolerance to TSHR does not occur in mice and other placental mammals and this could explain why GD is a purely human disease. In fact, in mice, tolerance to TSHR is very solid and investigators had to resort to the generation of TSHR -/mice [53], or to use very intense immunisation protocols to elicit a response to the TSHR [54]. This response only results in a very mod- erate thyroid lesion and minimally detectable effect on thyroid function and some degree of Graves' ophthalmopathy.…”