Review of Mouse Models of Graves' Disease and Orbitopathy-Novel Treatment by Induction of Tolerance

Abstract

Various approaches have been used to model human Graves' disease in mice, including transfected fibroblasts, and plasmid or adenoviral immunisations with the extracellular A subunit of the human thyrotropin receptor (TSHR). Some of these models were only observed for a short time period or were self-limiting. A long-term model for human Graves' disease was established in mice using continuing immunisations (4-weekly injections) with recombinant adenovirus expressing TSHR. Generation of TSHR binding cAMP-stimulatory antibodies, thyroid enlargement and alterations, elevated serum thyroxin levels, tachycardia and cardiac hypertrophy were maintained for at least 9 months in all Ad-TSHR-immunised mice. Here, we show that these mice suffer from orbitopathy, which was detected by serial orbital sectioning and histomorphometry. Attempts to treat established Graves' disease in preclinical mouse model studies have included small molecule allosteric antagonists and specific antagonist antibodies which were isolated from hypothyroid patients. In addition, novel peptides have been conceived which mimic the cylindrical loops of the TSHR leucine-rich repeat domain, in order to re-establish tolerance toward the antigen. Here, we show preliminary results that one set of these peptides improves or even cures all signs and symptoms of Graves' disease in mice after six consecutive monthly injections. First beneficial effects were observed 3-4 months after starting these therapies. In immunologically naïve mice, administration of the peptides did not induce any immune response.

Keywords: Autoimmunity; Graves’ disease; Peptides; Thyreotropin receptor; Tolerance.

Fig. 1

Histological investigation of orbital sections. a Representative macroscopic images. Representative images of coronary sections of a mouse orbita and neighbouring tissues. The sections were taken at defined distances from the mouse bregma. Interstitial connective tissue/fibrosis was then stained in green (Masson’s trichrome stain). For clarity, both HE-stained sections (left panels) and Masson’s stained sections (right panels) are shown next to each other. b Effect on digitised analysis of retroorbital tissue. The effects on severity of retro-orbital fibrosis were evaluated in histological sections. The measurements were carried out in immunised mice treated by either four weekly injections with control Ad-GFP (“mock-immunised”) or four weekly injections with Ad-TSHR (“Graves’ diesease”). N = 10 mice were investigated in each group. The mean total fibrosis volumes of each right and left orbita, as assessed by digitised image analysis of all sections, and consecutive integrations, are shown with SEM. Differences between groups were tested by ANOVA, *p < 0.05 compared to the TSHR-immunised group

Fig. 2

Antigen-specific peptides can induce peripheral tolerance. The figure shows that administration of peptides can induce peripheral T or B lymphocyte anergy if presented to antigen-presenting immune cells in the absence of a co-stimulatory signal

Fig. 3

a Time schedule of the currently on-going therapeutic study. The figure shows the time course of immunisations, of therapeutic peptide administrations and of measurements. b Schematic structure of the thyroid stimulating hormone (TSH) receptor. Peptides were derived from the loop structure of the leucine-rich repeat domain of the extracellular A subunit of the TSHR, as marked in blue colour

Fig. 4

Effect of novel peptides on anti-TSHR antibody titres. The effect of peptide therapy on time course of anti-TSHR titres, as measured by third generation ELISA. The measurements were carried out in Ad-TSHR-immunised mice treated by either four weekly injections with vehicle (“Graves’—no therapy”), or administrations of a peptide (1 mg/kg body weight—“Graves’ + peptide”). In addition, age-matched immunologically naïve mice were investigated (“healthy”). N = 10 mice were investigated in each group. Data are represented as mean ± SEM. Significance over time was tested by analysis of variance (ANOVA) of groups at given time points and controlled by ANOVA for repeated measurements within one group, followed by LSD post hoc testing. Asterisk indicates statistical significance (p < 0.05) compared to the TSHR-immunised group treated with only NaCl

Fig. 5

Effect of peptides on macroscopically measured thyroid size. The effects of peptide therapy on thyroid sizes were investigated at the end of the experiment. The measurements were carried out in Ad-TSHR-immunised mice treated by either four weekly injections with vehicle (“Graves’—no therapy”) or administrations of peptide (1 mg/kg body weight). In addition, age-matched immunologically naïve mice were investigated. N = 10 mice were investigated in each group. The mean thyroid sizes in mm³ are shown with SEM. Differences between groups were tested by AVOVA followed by post hoc LSD testing. Asterisk indicates statistical significance (p < 0.05) compared to the TSHR-immunised group treated with only NaCl

Fig. 6

Effect on serum thyroxin (T4) levels. The effects of peptide therapy on serum thyroxin levels were evaluated. The measurements were carried out in Ad-TSHR-immunised mice treated by either four weekly injections with vehicle (“Graves’—no therapy”) or of a peptide (1 mg/kg body weight), or in age-matched immunologically naïve mice. Data are represented as means ± SEM. N = 10 mice were investigated in each group. Significance over time was tested by analysis of variance (ANOVA) of groups at given time points and controlled by ANOVA for repeated measurements within one group, followed by LSD post hoc testing. *p < 0.05 and **p < 0.01, compared to the TSHR-immunised group treated with only NaCl

Fig. 7

Histological investigation of orbital sections and digitised analysis of retroorbital tissue: effects of novel peptides. The effects of peptide therapy on severity of retro-orbital fibrosis were evaluated in histological sections. The measurements were carried out in mice which had either received four weekly injections with vehicle (“Graves’—no therapy”) or of a peptide (1 mg/kg body weight), or in age-matched immunologically naïve mice. The mean total fibrosis volumes of each right and left orbita, as assessed by digitised image analysis of all sections, and consecutive integrations, are shown with SEM. N = 10 mice were investigated in each group. Differences between groups were tested by ANOVA, *p < 0.05 compared to the TSHR-immunised group treated with only NaCl. Also, results of TSHR-immunised mice differed significantly (p < 0.05) from healthy controls