Thyroid Hemiagenesis in a Thyroiditis Prone Mouse Strain.

BACKGROUND: Thyroid hemiagenesis, a rare congenital condition detected by ultrasound screening of the neck, is usually not manifested clinically in humans. This condition has been reported in mice with hypothyroidism associated with induced deficiency in paired box 8 and NK2 homeobox 1, sonic hedgehog, or T-box 1. Unexpectedly, we observed thyroid hemiagenesis in NOD.H2h4 mice, an unusual strain that spontaneously develops iodide enhanced thyroid autoimmunity but remains euthyroid. OBJECTIVES AND METHODS: First, to compare mice with thyroid hemiagenesis versus bilobed littermates for serum T4, autoantibodies to thyroglobulin (ELISA) and thyroid peroxidase (TPO; flow cytometry with eukaryotic cells expressing mouse TPO), gross anatomy, and thyroid histology; second, to estimate the percentage of mice with thyroid hemiagenesis in the NOD.H2h4 mice we have studied over 6 years. RESULTS: Thyroid hemiagenesis was observed in 3 of 1,025 NOD.H2h4 mice (2 females, 1 male; 0.3$). Two instances of hemiagenesis were in wild-type females and one in a transgenic male expressing the human TSHR A-subunit in the thyroid. Two mice had very large unilobed glands, as in some human cases with this condition. Thyroid lymphocytic infiltration, serum T4, and the levels of thyroid autoantibodies were similar in mice with thyroid hemiagenesis and bilobed littermates. CONCLUSIONS: Unlike hypothyroidism associated with hemiagenesis in transcription factor knockout mice, hemiagenesis in euthyroid NOD.H2h4 mice occurs spontaneously and is phenotypically similar to that occasionally observed in humans.

Variable Effects of Dietary Selenium in Mice That Spontaneously Develop a Spectrum of Thyroid Autoantibodies.

Selenium (Se) is a critical element in thyroid function, and variable dietary Se intake influences immunity. Consequently, dietary Se could influence development of thyroid autoimmunity and provide an adjunct to treat autoimmune thyroid dysfunction. Nonobese diabetic (NOD).H2h4 mice spontaneously develop autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO). This mouse strain expressing a human thyroid-stimulating hormone receptor (TSHR) A-subunit transgene in the thyroid also develops pathogenic TSHR autoantibodies. In this report, we investigated whether dietary Se influences these immune processes. Male and female wild-type and transgenic NOD.H2h4 mice were maintained on normal-, low-, or high-Se (0.1, 0, or 1.0 mg/kg) rodent diets. After 4 months, Se serum levels were extremely low or significantly increased on 0 or 1.0 mg/kg Se, respectively. Varying Se intake affected Tg antibody (TgAb) levels after 2 (but not 4) months; conversely, TPO antibody (TPOAb) levels were altered by dietary Se after 4 (but not 2) months. These data correspond to the earlier development of TgAb than TPOAb in NOD.H2h4 mice. In males, TgAb levels were enhanced by high Se and in females by low Se intake. Se intake had no effect on pathogenic TSHR autoantibodies in TSHR transgenic NOD.H2h4 females. In conclusion, in susceptible NOD.H2h4 mice, we found no evidence that a higher dietary Se intake ameliorates thyroid autoimmunity by reducing autoantibodies to Tg, TPO, or the TSHR. Instead, our finding that low dietary Se potentiates the development of autoantibodies to Tg and TPO in females is consistent with reports in humans of an increased prevalence of autoimmune thyroiditis in low-Se regions.

Genes Outside the Major Histocompatibility Complex Locus Are Linked to the Development of Thyroid Autoantibodies and Thyroiditis in NOD.H2h4 Mice.

Thyroiditis and autoantibodies to thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) develop spontaneously in NOD.H2h4 mice, a phenotype enhanced by dietary iodine. NOD.H2h4 mice were derived by introducing the major histocompatibility class (MHC) molecule I-Ak from B10.A(4R) mice to nonobese diabetic (NOD) mice. Apart from I-Ak, the genes responsible for the NOD.H2h4 phenotype are unknown. Extending serendipitous observations from crossing BALB/c to NOD.H2h4 mice, thyroid autoimmunity was investigated in both genders of the F1, F2, and the second-generation backcross of F1 to NOD.H2h4 (N2). Medium-density linkage analysis was performed on thyroid autoimmunity traits in F2 and N2 progeny. TgAb develop before TPOAb and were measured after 8 and 16 weeks of iodide exposure; TPOAb and thyroiditis were studied at 16 weeks. TgAb, TPOAb, and thyroiditis, absent in BALB/c and F1 mice, developed in most NOD.H2h4 and in more N2 than F2 progeny. No linkages were observed in F2 progeny, probably because of the small number of autoantibody-positive mice. In N2 progeny (equal numbers of males and females), a chromosome 17 locus is linked to thyroiditis and TgAb and is suggestively linked to TPOAb. This locus includes MHC region genes from B10.A(4R) mice (such as I-Ak and Tnf, the latter involved in thyrocyte apoptosis) and genes from NOD mice such as Satb1, which most likely plays a role in immune tolerance. In conclusion, MHC and non-MHC genes, encoded within the chromosome 17 locus from both B10.A(4R) and NOD strains, are most likely responsible for the Hashimoto disease-like phenotype of NOD.H2h4 mice.

Critical Differences between Induced and Spontaneous Mouse Models of Graves' Disease with Implications for Antigen-Specific Immunotherapy in Humans.

Graves' hyperthyroidism, a common autoimmune disease caused by pathogenic autoantibodies to the thyrotropin (TSH) receptor (TSHR), can be treated but not cured. This single autoantigenic target makes Graves' disease a prime candidate for Ag-specific immunotherapy. Previously, in an induced mouse model, injecting TSHR A-subunit protein attenuated hyperthyroidism by diverting pathogenic TSHR Abs to a nonfunctional variety. In this study, we explored the possibility of a similar diversion in a mouse model that spontaneously develops pathogenic TSHR autoantibodies, NOD.H2h4 mice with the human (h) TSHR (hTSHR) A-subunit transgene expressed in the thyroid and (shown in this article) the thymus. We hypothesized that such diversion would occur after injection of "inactive" hTSHR A-subunit protein recognized only by nonpathogenic (not pathogenic) TSHR Abs. Surprisingly, rather than attenuating the pre-existing pathogenic TSHR level, in TSHR/NOD.H2h4 mice inactive hTSHR Ag injected without adjuvant enhanced the levels of pathogenic TSH-binding inhibition and thyroid-stimulating Abs, as well as nonpathogenic Abs detected by ELISA. This effect was TSHR specific because spontaneously occurring autoantibodies to thyroglobulin and thyroid peroxidase were unaffected. As controls, nontransgenic NOD.H2h4 mice similarly injected with inactive hTSHR A-subunit protein unexpectedly developed TSHR Abs, but only of the nonpathogenic variety detected by ELISA. Our observations highlight critical differences between induced and spontaneous mouse models of Graves' disease with implications for potential immunotherapy in humans. In hTSHR/NOD.H2h4 mice with ongoing disease, injecting inactive hTSHR A-subunit protein fails to divert the autoantibody response to a nonpathogenic form. Indeed, such therapy is likely to enhance pathogenic Ab production and exacerbate Graves' disease in humans.

A unique mouse strain that develops spontaneous, iodine-accelerated, pathogenic antibodies to the human thyrotrophin receptor.

Abs that stimulate the thyrotropin receptor (TSHR), the cause of Graves' hyperthyroidism, only develop in humans. TSHR Abs can be induced in mice by immunization, but studying pathogenesis and therapeutic intervention requires a model without immunization. Spontaneous, iodine-accelerated, thyroid autoimmunity develops in NOD.H2(h4) mice associated with thyroglobulin and thyroid-peroxidase, but not TSHR, Abs. We hypothesized that transferring the human TSHR A-subunit to NOD.H2(h4) mice would result in loss of tolerance to this protein. BALB/c human TSHR A-subunit mice were bred to NOD.H2(h4) mice, and transgenic offspring were repeatedly backcrossed to NOD.H2(h4) mice. All offspring developed Abs to thyroglobulin and thyroid-peroxidase. However, only TSHR-transgenic NOD.H2(h4) mice (TSHR/NOD.H2(h4)) developed pathogenic TSHR Abs as detected using clinical Graves' disease assays. As in humans, TSHR/NOD.H2(h4) female mice were more prone than male mice to developing pathogenic TSHR Abs. Fortunately, in view of the confounding effect of excess thyroid hormone on immune responses, spontaneously arising pathogenic human TSHR Abs cross-react poorly with the mouse TSHR and do not cause thyrotoxicosis. In summary, the TSHR/NOD.H2(h4) mouse strain develops spontaneous, iodine-accelerated, pathogenic TSHR Abs in female mice, providing a unique model to investigate disease pathogenesis and test novel TSHR Ag-specific immunotherapies aimed at curing Graves' disease in humans.

Immunoglobulin heavy chain variable region and major histocompatibility region genes are linked to induced graves' disease in females from two very large families of recombinant inbred mice.

Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans.

Behaviorally specific versus non-specific suppression of accumbens shell-mediated feeding by ipsilateral versus bilateral inhibition of the lateral hypothalamus.

The nucleus accumbens shell (AcbSh) and lateral hypothalamus (LH) are linked in the control of food intake. Pharmacological inhibition of the LH may block AcbSh-elicited feeding, but the behavioral phenotype associated with this feeding suppression is unknown. To examine this phenotype, adult male Sprague-Dawley rats were implanted with three cannulas - one unilaterally in the AcbSh and two bilaterally in the LH - to allow for central drug injections. The AcbSh received injections of the AMPA receptor antagonist DNQX or the GABAA receptor agonist muscimol, while the LH received injections of the NMDA receptor antagonist D-AP5 or muscimol. Eating, drinking, grooming, locomotion, quiescence, and sleeping behaviors were measured every minute for 60 min post-injection. From these observational data, feeding bout durations, feeding frequency, and latency to feed were determined. AcbSh muscimol or DNQX increased food intake by increasing feeding bout durations and frequency and decreasing latency to feed. D-AP5 or muscimol, injected into the LH bilaterally or ipsilateral to the AcbSh injection, reversed these AcbSh-mediated effects. Though bilateral LH D-AP5 or muscimol injections blocked feeding responses, they also hastened onset of sleep. In contrast, ipsilateral LH D-AP5 or muscimol injections suppressed AcbSh-mediated feeding behaviors without substantially altering sleeping or other behaviors. These results suggest bilateral LH inhibition via NMDA receptor blockade or GABAA receptor activation produces behavioral effects that might indirectly suppress feeding, but ipsilateral LH inhibition through these receptors suppresses AcbSh AMPA and GABAA receptor-mediated feeding specifically. This evidence strengthens the concept of a feeding-specific association between these regions.