Linsitinib, an IGF-1R inhibitor, attenuates disease development and progression in a model of thyroid eye disease.

Introduction: Graves' disease (GD) is an autoimmune disorder caused by autoantibodies against the thyroid stimulating hormone receptor (TSHR) leading to overstimulation of the thyroid gland. Thyroid eye disease (TED) is the most common extra thyroidal manifestation of GD. Therapeutic options to treat TED are very limited and novel treatments need to be developed. In the present study we investigated the effect of linsitinib, a dual small-molecule kinase inhibitor of the insulin-like growth factor 1 receptor (IGF-1R) and the Insulin receptor (IR) on the disease outcome of GD and TED. Methods: Linsitinib was administered orally for four weeks with therapy initiating in either the early ("active") or the late ("chronic") phases of the disease. In the thyroid and the orbit, autoimmune hyperthyroidism and orbitopathy were analyzed serologically (total anti-TSHR binding antibodies, stimulating anti TSHR antibodies, total T4 levels), immunohistochemically (H&E-, CD3-, TNFa- and Sirius red staining) and with immunofluorescence (F4/80 staining). An MRI was performed to quantify in vivo tissue remodeling inside the orbit. Results: Linsitinib prevented autoimmune hyperthyroidism in the early state of the disease, by reducing morphological changes indicative for hyperthyroidism and blocking T-cell infiltration, visualized by CD3 staining. In the late state of the disease linsitinib had its main effect in the orbit. Linsitinib reduced immune infiltration of T-cells (CD3 staining) and macrophages (F4/80 and TNFa staining) in the orbita in experimental GD suggesting an additional, direct effect of linsitinib on the autoimmune response. In addition, treatment with linsitinib normalized the amount of brown adipose tissue in both the early and late group. An in vivo MRI of the late group was performed and revealed a marked decrease of inflammation, visualized by 19F MR imaging, significant reduction of existing muscle edema and formation of brown adipose tissue. Conclusion: Here, we demonstrate that linsitinib effectively prevents development and progression of thyroid eye disease in an experimental murine model for Graves' disease. Linsitinib improved the total disease outcome, indicating the clinical significance of the findings and providing a path to therapeutic intervention of Graves' Disease. Our data support the use of linsitinib as a novel treatment for thyroid eye disease.

Predicting the Course of Graves' Orbitopathy Using Serially Measured TSH-Receptor Autoantibodies by Automated Binding Immunoassays and the Functional Bioassay.

The aim of the study was to investigate the use of serial measurements of TSH-receptor autoantibodies (TRAb) with the newest available assay technology to predict the course of Graves' Orbitopathy (GO) during the first 24 months from disease onset. Serial serum samples from patients with GO (103 mild/135 severe) were collected between 2007 and 2017 and retrospectively analyzed. The course of GO were classified into mild/severe 12 months after manifestation (severe: NOSPECS≥5; mild<5). TRAb were measured with automated binding immunoassays (IU/l): TRAb Elecsys (Cobas, Roche), TRAb bridge assay (IMMULITE, Siemens), and a cell-based bioassay (percent of specimen to reference ratio - SRR%) (Thyretain, Quidel). Variable cut off levels of measured TRAb were calculated at specificity of 90% from receiver operator curve (ROC) analysis for several timepoints during the course of GO. To select one: 5-8 months after first GO symptoms, which is the timepoint for usual referals for treatment mild course could be predicted at cut offs of 1.5 IU/l (Elecsys), 0.8 IU/l (Immulite) and 402% SRR (Thyretain) and the risc of severe course has to be anticipated if TRAb are above 11.6 IU/l (Elecsys), 6.5 IU/l (Thyretain), and 714% SRR (Thyretain). The Thyretain bioassay showed the highest diagnostic sensitivity (using the commercial cut off's) over the entire follow up period. TRAb measurements during the 24-month follow up of GO provide added value to the GO clinical activity and severity scores and should be used especially in the event of an unclear decision-taking situation with regard to therapy.

Predicting the Relapse of Hyperthyroidism in Treated Graves' Disease with Orbitopathy by Serial Measurements of TSH-Receptor Autoantibodies.

The aim of this study was to investigate the potential of the new TSH-receptor antibody (TRAb) assays to predict remission or relapse of hyperthyroidism in patients with Graves' disease (GD) and Graves' orbitopathy (GO). TRAbs were measured retrospectively in sera from a cohort of GD patients with GO (n=117; remission n=38 and relapse n=79-Essen GO biobank) with automated binding immunoassays: TRAb Elecsys (Cobas Roche) and TRAb bridge assay (IMMULITE, Siemens), and the TSAb (thyroid stimulating Ab) cell-based bioassay (Thyretain, Quidel Corp.). To identify relapse risk/remission of hyperthyroidism patients were followed up at least 10 months after the end of antithyroid drug therapy (ATD) therapy. ROC plot analysis was performed to calculate cut-off levels of TRAb and TSAb for prediction of relapse and remission of hyperthyroidism. Cut-off serum levels are provided for timepoints around 3, 6, 10, and 15 months after the beginning of ATD. Repeated measurements of TRAb increase the rate of relapses predictions to 60% (Elecsys), 70% (IMMULITE), and 55% (Thyretain). Patients with remission have consistently TRAb levels below the cut off for relapse in repeated measurements. The cell-based bioassay was the most sensitive - and continued to be positive during follow up [at 15 months: 90% vs. 70% (IMMULITE) and 65% (Elecsys)]. Identification of relapsing hyperthyroidism is possible with automated immunoassays and cell-based bioassay especially with serial TRAb measurements during the course of ATD therapy. Patient who need eye surgery may profit from an early decision towards definitive treatment.

Modulating gut microbiota in a mouse model of Graves' orbitopathy and its impact on induced disease.

BACKGROUND: Graves' disease (GD) is an autoimmune condition in which autoantibodies to the thyrotropin receptor (TSHR) cause hyperthyroidism. About 50% of GD patients also have Graves' orbitopathy (GO), an intractable disease in which expansion of the orbital contents causes diplopia, proptosis and even blindness. Murine models of GD/GO, developed in different centres, demonstrated significant variation in gut microbiota composition which correlated with TSHR-induced disease heterogeneity. To investigate whether correlation indicates causation, we modified the gut microbiota to determine whether it has a role in thyroid autoimmunity. Female BALB/c mice were treated with either vancomycin, probiotic bacteria, human fecal material transfer (hFMT) from patients with severe GO or ddH2O from birth to immunization with TSHR-A subunit or beta-galactosidase (ßgal; age ~ 6 weeks). Incidence and severity of GD (TSHR autoantibodies, thyroid histology, thyroxine level) and GO (orbital fat and muscle histology), lymphocyte phenotype, cytokine profile and gut microbiota were analysed at sacrifice (~ 22 weeks). RESULTS: In ddH2O-TSHR mice, 84% had pathological autoantibodies, 67% elevated thyroxine, 77% hyperplastic thyroids and 70% orbital pathology. Firmicutes were increased, and Bacteroidetes reduced relative to ddH2O-ßgal; CCL5 was increased. The random forest algorithm at the genus level predicted vancomycin treatment with 100% accuracy but 74% and 70% for hFMT and probiotic, respectively. Vancomycin significantly reduced gut microbiota richness and diversity compared with all other groups; the incidence and severity of both GD and GO also decreased; reduced orbital pathology correlated positively with Akkermansia spp. whilst IL-4 levels increased. Mice receiving hFMT initially inherited their GO donors' microbiota, and the severity of induced GD increased, as did the orbital brown adipose tissue volume in TSHR mice. Furthermore, genus Bacteroides, which is reduced in GD patients, was significantly increased by vancomycin but reduced in hFMT-treated mice. Probiotic treatment significantly increased CD25+ Treg cells in orbital draining lymph nodes but exacerbated induced autoimmune hyperthyroidism and GO. CONCLUSIONS: These results strongly support a role for the gut microbiota in TSHR-induced disease. Whilst changes to the gut microbiota have a profound effect on quantifiable GD endocrine and immune factors, the impact on GO cellular changes is more nuanced. The findings have translational potential for novel, improved treatments. Video abstract.

Structural Studies of Thyroid Peroxidase Show the Monomer Interacting With Autoantibodies in Thyroid Autoimmune Disease.

Thyroid peroxidase (TPO) is a critical membrane-bound enzyme involved in the biosynthesis of multiple thyroid hormones, and is a major autoantigen in autoimmune thyroid diseases such as destructive (Hashimoto) thyroiditis. Here we report the biophysical and structural characterization of a novel TPO construct containing only the ectodomain of TPO and lacking the propeptide. The construct was enzymatically active and able to bind the patient-derived TR1.9 autoantibody. Analytical ultracentrifugation data suggest that TPO can exist as both a monomer and a dimer. Combined with negative stain electron microscopy and molecular dynamics simulations, these data show that the TR1.9 autoantibody preferentially binds the TPO monomer, revealing conformational changes that bring together previously disparate residues into a continuous epitope. In addition to providing plausible structural models of a TPO-autoantibody complex, this study provides validated TPO constructs that will facilitate further characterization, and advances our understanding of the structural, functional, and antigenic characteristics of TPO, an autoantigen implicated in some of the most common autoimmune diseases.

Thyrotropin, but Not Thyroid-Stimulating Antibodies, Induces Biphasic Regulation of Gene Expression in Human Thyrocytes.

Background: Thyrotropin (TSH) and thyroid-stimulating antibodies (TSAbs) activate TSH receptor (TSHR) signaling by binding to its extracellular domain. TSHR signaling has been studied extensively in animal thyrocytes and in engineered cell lines, and differences in signaling have been observed in different cell systems. We, therefore, decided to characterize and compare TSHR signaling mediated by TSH and monoclonal TSAbs in human thyrocytes in primary culture. Methods: We used quantitative reverse transcription-polymerase chain reaction to measure mRNA levels of thyroid-specific genes thyroglobulin (TG), thyroperoxidase (TPO), iodothyronine deiodinase type 2 (DIO2), sodium-iodide symporter (NIS), and TSHR after stimulation by TSH or two monoclonal TSAbs, KSAb1 and M22. We also compared secreted TG protein after TSHR activation by TSH and TSAbs using an enzyme-linked immunosorbent assay. TSHR cell surface expression was determined using fluorescence activated cell sorting (FACS). Results: We found that TSH at low doses increases and at high doses (>1 mU/mL) decreases levels of gene expression for TSHR, TG, TPO, NIS, and DIO2. The biphasic effect of TSH on signaling was not caused by downregulation of cell surface TSHRs. This bell-shaped biphasic dose-response curve has been termed an inverted U-shaped dose-response curve (IUDRC). An IUDRC was also found for TSH-induced regulation of TG secretion. In contrast, KSAb1- and M22-induced regulation of TSHR, TG, TPO, NIS, and DIO2 gene expression, and secreted TG followed a monotonic dose-response curve that plateaus at high doses of activating antibody. Conclusions: Our data demonstrate that the physiological activation of TSHRs by TSH in primary cultures of human thyrocytes is characterized by a regulatory mechanism that may inhibit thyrocyte overstimulation. In contrast, TSAbs do not exhibit biphasic regulation. Although KSAb1 and M22 may not be representative of all TSAbs found in patients with Graves' disease, we suggest that persistent robust stimulation of TSHRs by TSAbs, unrelieved by a decrease at high TSAb levels, fosters chronic stimulation of thyrocytes in Graves' hyperthyroidism.

Thyroid Peroxidase Revisited - What's New?

Thyroid peroxidase (TPO) is an enzyme that participates in thyroid hormone biosynthesis. TPO is also a major autoantigen in autoimmune thyroid diseases (AITD). In this review, we summarize the latest developments in the field of TPO research. We present the current understanding of immunodominant serologic determinants, frequency of TPO-specific autoantibodies in the population, as well as genetic and environmental factors contributing to their development. Moreover, we report recent progress in the clinical utilities of TPO autoantibody testing, including thyroid dysfunctions and extra-thyroidal disorders.

Human placenta-derived mesenchymal stem cells ameliorate orbital adipogenesis in female mice models of Graves' ophthalmopathy.

BACKGROUND: Graves' ophthalmopathy (GO) is a complication of Graves' disease (GD), in which orbital connective tissues become inflamed and increase in volume and orbital fibroblasts within the orbital fat and extraocular muscles differentiate into adipocytes in vitro when stimulated by hormones, several cytokines, and growth factors including TSH, IGF-1, IL-1, interferon γ, and platelet-derived growth factor. Human placental mesenchymal stem cells (hPMSCs) have immunomodulatory effects in disease pathogenesis. Although a number of studies have reported that hPMSCs can elicit therapeutic effects, these are not sufficient. Therefore, we constructed a GO animal model in order to find out the hPMSCs recovery effect. METHODS: We investigated their anti-adipogenic effects in in vitro cultures of orbital fibroblasts established from GO patients. Primary orbital fibroblasts were exposed to differentiation medium for 10 days. After being co-cultured with hPMSCs, the characteristics of orbital fibroblast were determined by Oil Red O stain and real-time PCR. Then, we explored the in vivo regulatory effects of hPMSCs in an experimental mouse model of GO. We developed the GO mouse model using immunization by leg muscle electroporation of pTriEx1.1Neo-hTSHR A-subunit plasmid. Human PMSC injection was performed into the left orbit. We also analyzed the effects of hPMSCs in the GO animal model. RESULT: We found that hPMSCs inhibited a lipid accumulation and activated factors, such as ADIPONECTIN, PPARγ, C/EBPα, and TGFß2 genes in adipogenesis-induced primary orbital fibroblasts from GO patients. Moreover, hPMSCs were highly effective at ameliorating adipogenesis in the orbital tissue of the model. CONCLUSION: These data indicate that hPMSCs recover pathogenic activation of orbital fibroblasts in animals undergoing experimental GO and confirm the feasibility of applying hPMSCs as a novel treatment for GO patients.

Arrestin-ß-1 Physically Scaffolds TSH and IGF1 Receptors to Enable Crosstalk.

Endogenously expressed TSH receptors (TSHRs) on orbital fibroblasts of patients with Graves ophthalmopathy (GO) use crosstalk with IGF1 receptors (IGF1R) to synergistically stimulate secretion of hyaluronan (HA), a major component of GO pathology. We previously showed crosstalk occurred upstream of mitogen-activated protein kinase (ERK) phosphorylation. Because other G protein-coupled receptors engage arrestin-ß-1 (ARRB1) and ERK, we tested whether ARRB1 was a necessary component of TSHR/IGF1R crosstalk. HA secretion was stimulated by the TSHR-stimulating monoclonal antibodies M22 and KSAb1, or immunoglobulins from patients with GO (GO-Igs). Treatment with M22, as previously shown, resulted in biphasic dose-response stimulation of HA secretion. The high-potency phase was IGF1R dependent, and the low-potency phase was partly IGF1R independent. KSAb1 produced a monophasic dose-response stimulation of HA secretion, whose potency was lowered >20-fold after IGF1R knockdown. ARRB1 knockdown abolished M22's high-potency phase and lowered KSAb1's potency and efficacy. ARRB1 knockdown inhibited GO-Ig stimulation of HA secretion and of ERK phosphorylation. Last, ARRB1 was shown to be necessary for TSHR/IGF1R proximity. In contrast, ARRB2 knockdowns did not show these effects. Thus, TSHR must neighbor IGF1R for crosstalk in GO fibroblasts to occur, and this depends on ARRB1 acting as a scaffold. Similar scaffolding of TSHR and IGF1R by ARRB1 was found in human osteoblast-like cells and human thyrocytes. These findings support a model of TSHR/IGF1R crosstalk that may be a general mechanism for G-protein-coupled receptor/receptor tyrosine kinase crosstalk dependent on ARRB1.

Graves' orbitopathy occurs sex-independently in an autoimmune hyperthyroid mouse model.

Graves' orbitopathy (GO) is the most common extra thyroidal complication of Graves' disease (GD) and occurs predominantly in women but more severe in men. The reason for this effect of gender on GO is unknown. Herein we studied the manifestation of GO in both sexes of an induced mouse model in absence of additional risk factors present in patients like advanced age, genetic variabilities or smoking. Male and female mice were immunized with human TSHR A-subunit encoding plasmid. Both sexes comparably developed autoimmune hyperthyroidism characterized by TSHR stimulating autoantibodies, elevated T4 values, hyperplastic thyroids and hearts. Autoimmune mice developed inflammatory eye symptoms and proptosis, although males earlier than females. Serial in vivo 1H/19F-magnetic resonance imaging revealed elevated inflammatory infiltration, increased fat volume and glycosaminoglycan deposition in orbits of both sexes but most significantly in female mice. Histologically, infiltration of T-cells, extension of brown fat and overall collagen deposition were characteristics of GO in male mice. In contrast, female mice developed predominately macrophage infiltration in muscle and connective tissue, and muscle hypertrophy. Apart from sex-dependent variabilities in pathogenesis, disease classification revealed minor sex-differences in incidence and total outcome. In conclusion, sex does not predispose for autoimmune hyperthyroidism and associated GO.

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

Experimental models of hyperthyroid Graves' disease (GD) and Graves' orbitopathy (GO) are efficiently developed by genetic immunisation by electroporation with human thyrotropin hormone receptor (hTSHR) A-subunit plasmid in female BALB/c (H-2d) mice. We investigated susceptibility in C57BL/6 J (H-2b) animals to allow studies on disease mechanisms in transgenic and immune response gene knock-out mice. Higher numbers of female C57BL/6 J were positive for pathogenic thyroid stimulating antibodies, but induced hyperthyroidism remained at a low frequency compared to BALB/c animals. Assessment of hTSHR specific T cells showed reduced proliferation in C57BL/6 J animals accompanied with anti-inflammatory IL-10, with less pro-inflammatory IFN-γ compared to BALB/c. Whilst the orbital tissue from immune BALB/c mice showed inflammation and adipogenesis, in contrast C57BL/6 J animals showed normal pathology. We characterised the gut microbiota using 16 S ribosomal RNA gene sequencing to explore its possible pathogenic role in the model. Despite being housed under identical conditions, we observed significantly different organisation of the microbiota (beta-diversity) in the two strains. Taxonomic differences were also noted, with C57BL/6 J showing an enrichment of Operational Taxonomic Units (OTUs) belonging to the Paludibacter and Allobaculum, followed by Limibacter, Anaerophaga and Ureaplasma genera. A higher number of genera significantly correlating with clinical features was observed in C57BL/6 J compared to BALB/c; for example, Limibacter OTUs correlated negatively with thyroid-stimulating antibodies in C57BL/6 J mice. Thus, our data suggest gut microbiota may play a pivotal immunomodulatory role that differentiates the thyroid function and orbital pathology outcome in these two inbred strains undergoing experimental GO.

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

OBJECTIVES: 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. The interrelated pathological findings in the thyroid glands of Graves disease (GD) that explain the core changes classically include diffuse follicular hyperplasia and multifocal mild lymphocytic infiltrate. However, the relative contributions of different thyroid tissue components (colloid, follicular cells, and stroma) have not been previously evaluated. In this study, we characterize the thyroid gland of an experimental mouse model of autoimmune GD. Our objective was to define the relative contribution of the different thyroid tissue components to the pathology of glands in the experimental model. METHODS: Mice were immunized with human TSHR A-subunit plasmid. Antibodies induced to human TSHR were pathogenic in vivo due to their cross-reactivity to mouse TSHR. RESULTS: Autoimmune thyroid disease in the model was characterized by histopathology of hyperplastic glands with large follicular cells. Further examination of thyroid glands of immunized animals revealed a significantly increased follicular area and follicle/stroma ratio, morphometrically correlated with a noninflammatory follicular hyperplasia/hypertrophy. The increased follicle/stroma ratio was the most relevant morphometrically variable summarizing the pathological changes for screening purposes. CONCLUSION: GD thyroid glands are enlarged and characterized by a noninflammatory diffuse follicular cell hyperplasia/hypertrophy and a significant increase in the follicles with an increased follicle/stroma ratio. Overall, this mouse model is a faithful model of an early hyperthyroid status of GD (diffuse glandular involvement and follicular expansion).

Multimodal assessment of orbital immune cell infiltration and tissue remodeling during development of graves disease by 1 H19 F MRI.

PURPOSE: To evaluate key molecular and cellular features of Graves orbitopathy (GO) by simultaneous monitoring of alterations in morphology, inflammatory patterns, and tissue remodeling. METHODS: To this end, we utilized a murine model of GO induced by immunization with a human thyroid-stimulating hormone receptor A-subunit plasmid. Altogether, 52 mice were used: 27 GOs and 25 controls (Ctrl) immunized with ß-galactasidose plasmid. From these, 17 GO and 12 Ctrl mice were subjected to multimodal MRI at 9.4T, whereas 23 mice only underwent histology. Beyond anatomical hydrogen-1 (1 H) MRI, we employed transverse relaxation time (T2 ) mapping for visualization of edema, chemical exchange saturation transfer (CEST) for detection of hyaluronan, and fluorine-19 (19 F) MRI for tracking of in situ-labeled immune cells after intravenous injection of perfluorcarbons (PFCs). RESULTS: 1 H/19 F MRI demonstrated substantial infiltration of PFC-loaded immune cells in peri and retro-orbital regions of GO mice, whereas healthy Ctrls showed only minor 19 F signals. In parallel, T2 mapping indicated onset of edema in periorbital tissue and adjacent ocular glands (P = 0.038/0.017), which were associated with enhanced orbital CEST signals in GO mice (P = 0.031). Concomitantly, a moderate expansion of retrobulbar fat (P = 0.029) was apparent; however, no signs for extraocular myopathy were detectable. 19 F MRI-based visualization of orbital inflammation exhibited the highest significance level to discriminate between GO and Ctrl mice (P = 0.006) and showed the best correlation with the clinical score (P = 0.0007). CONCLUSION: The present approach permits the comprehensive characterization of orbital tissue and holds the potential for accurate GO diagnosis in the clinical setting. Magn Reson Med 80:711-718, 2018. © 2018 International Society for Magnetic Resonance in Medicine.

De novo triiodothyronine formation from thyrocytes activated by thyroid-stimulating hormone.

The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating hormone receptors (TSHRs), patients develop a syndrome of relative T3 toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4 We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibody. We present data suggesting that TSH-stimulated TG phosphorylation contributes to enhanced de novo T3 formation. These effects were reversed within a few days after removal of the hyperstimulating conditions. Indeed, direct exposure of PCCL3 cells to human serum from two patients with Graves' disease, but not control sera, led to secretion of TG with an increased intrinsic ability to form T3 upon in vitro iodination. Furthermore, TG secreted from human thyrocyte cultures hyperstimulated with TSH also showed an increased intrinsic ability to form T3 Our data support the hypothesis that TG processing in the secretory pathway of TSHR-hyperstimulated thyrocytes alters the structure of the iodination substrate in a way that enhances de novo T3 formation, contributing to the relative T3 toxicosis of Graves' disease.

Pathogenic Phenotype of Adipogenesis and Hyaluronan in Orbital Fibroblasts From Female Graves' Orbitopathy Mouse Model.

A mouse model of Graves' orbitopathy (GO) induced by genetic immunization of human TSH receptor (TSHR) A-subunit encoding plasmid has recently been established. The orbital pathology was characterized by adipogenesis, myopathy and fibrosis. Human orbital fibroblasts (OFs) express TSHR and IGF-1 receptor (IGF-1R) and are considered to be pathogenic in GO. We established conditions for growing ex vivo cultures of mouse OFs (mOFs) from orbital tissue of animals undergoing GO and controls. Early passage mOFs showed characteristic fibroblast morphology and expressed mesenchymal stem cell markers including a strong expression of CD90.2 and CD40, whereas display of CD73 and all other leucocyte markers was uniformly absent. Importantly, OFs derived from GO mice expressed elevated levels of TSHR and IGF-1R and showed enhanced adipogensis compared with controls. Activation of TSHR in mOFs from GO animals with TSH, monoclonal thyroid-stimulating antibody M22, or stimulation of IGF-1R with IGF-1-induced hyaluronan secretion to significantly elevated levels compared with control animals. Hyaluronan synthase 2 was more abundant in OFs derived from GO mice. In conclusion, mOFs established from GO model recapitulate the pathogenicity of human OFs from GO patients by their increased propensity for adipogenesis and hyaluronan production leading to disease activity. To our knowledge, this is the first report to show mOFs from the preclinical GO model have pathogenic properties that will aid in understanding the molecular and genetic changes during progression to adipogenesis and hyaluronan deposition to provide new insights into GO pathogenesis.

Comparative Assessment of Female Mouse Model of Graves' Orbitopathy Under Different Environments, Accompanied by Proinflammatory Cytokine and T-Cell Responses to Thyrotropin Hormone Receptor Antigen.

We recently described a preclinical model of Graves' orbitopathy (GO), induced by genetic immunization of eukaryotic expression plasmid encoding human TSH receptor (TSHR) A-subunit by muscle electroporation in female BALB/c mice. The onset of orbital pathology is characterized by muscle inflammation, adipogenesis, and fibrosis. Animal models of autoimmunity are influenced by their environmental exposures. This follow-up study was undertaken to investigate the development of experimental GO in 2 different locations, run in parallel under comparable housing conditions. Functional antibodies to TSHR were induced in TSHR A-subunit plasmid-immunized animals, and antibodies to IGF-1 receptor α-subunit were also present, whereas control animals were negative in both locations. Splenic T cells from TSHR A-subunit primed animals undergoing GO in both locations showed proliferative responses to purified TSHR antigen and secreted interferon-γ, IL-10, IL-6, and TNF-α cytokines. Histopathological evaluation showed orbital tissue damage in mice undergoing GO, manifest by adipogenesis, fibrosis, and muscle damage with classic signs of myopathy. Although no inflammatory infiltrate was observed in orbital tissue in either location, the appearances were consistent with a "hit-and-run" immune-mediated inflammatory event. A statistically significant increase of cumulative incidence of orbital pathology when compared with control animals was shown for both locations, confirming onset of orbital dysimmune myopathy. Our findings confirm expansion of the model in different environments, accompanied with increased prevalence of T cell-derived proinflammatory cytokines, with relevance for pathogenesis. Wider availability of the model makes it suitable for mechanistic studies into pathogenesis and undertaking of novel therapeutic approaches.

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity.

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto's disease--the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The "trans" model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the "cis" model favours it slightly over the "trans" model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.

Preclinical models of Graves' disease and associated secondary complications.

Autoimmune thyroid disease is the most common organ-specific autoimmune disorder which consists of two opposing clinical syndromes, Hashimoto's thyroiditis and Graves' (hyperthyroidism) disease. Graves' disease is characterized by goiter, hyperthyroidism, and the orbital complication known as Graves' orbitopathy (GO), or thyroid eye disease. The hyperthyroidism in Graves' disease is caused by stimulation of function of thyrotropin hormone receptor (TSHR), resulting from the production of agonist antibodies to the receptor. A variety of induced mouse models of Graves' disease have been developed over the past two decades, with some reproducible models leading to high disease incidence of autoimmune hyperthyroidism. However, none of the models show any signs of the orbital manifestation of GO. We have recently developed an experimental mouse model of GO induced by immunization of the plasmid encoded ligand binding domain of human TSHR cDNA by close field electroporation that recapitulates the orbital pathology in GO. As in human GO patients, immune mice with hyperthyroid or hypothyroid disease induced by anti-TSHR antibodies exhibited orbital pathology and chemosis, characterized by inflammation of orbital muscles and extensive adipogenesis leading to expansion of the orbital retrobulbar space. Magnetic resonance imaging of the head region in immune mice showed a significant expansion of the orbital space, concurrent with proptosis. This review discusses the different strategies for developing mouse models in Graves' disease, with a particular focus on GO. Furthermore, it outlines how this new model will facilitate molecular investigations into pathophysiology of the orbital disease and evaluation of new therapeutic interventions.

A redundant role of human thyroid peroxidase propeptide for cellular, enzymatic, and immunological activity.

BACKGROUND: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. METHODS: An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPOΔpro; amino acid residues 21-108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. RESULTS: The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro was enzymatically active and recognized by patients' autoantibodies and a panel of epitope-specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPOΔpro was also maintained. Molecular modeling and dynamics simulations were consistent with these observations. CONCLUSIONS: Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of TPOΔpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and importantly is recognized by patients' autoantibodies, is a key advance for purification of substantial quantities of homogeneous preparation of TPO for crystallization, structural, and immunological studies.