Human TSH Receptor Research Articles

The immune response formed by the introduction of a DNA vector containing a cDNA fragment of the human thyroid-stimulating hormone receptor gene into transgenic mice

The number of patients with autoimmune thyroid diseases (Graves’disease, Hashimoto’sthyroiditis) is increasing globally. The most important part in the diagnosis of Graves’ disease (GD) is the detection of autoantibodies to the thyrotropin receptor (TSHR) in Graves’ patients’ sera. For the differential diagnosis of antibodies to thyroid antigens, it is promising to use tests based on monoclonal antibodies to TSHR, which can be obtained not only as a result of immunization with native or recombinant TSHR protein, but also through DNA immunization with genetically engineered constructs containing fragments of the TSHR gene. Based on mRNA we isolated from the thyroid tissue in GD, a number of fragments of the thyrotropin receptor gene were cloned, suitable for DNA immunization of animals. The purpose of this work is to evaluate the immunogenic properties of one of the constructed vectors, pVAX1-TSHR (1160), in a mouse model. The successful inclusion of the extracellular domain gene fragment of the human TSHR (1160), which was transfected into CHO cells as a part of the pVAX1 vector was confirmed by immunoblotting and ELISA. The immune response formed to the injection of the pVAX1 vector into BALB/c mice, containing a fragment of the human TSHR gene, was detected in different versions of ELISA. Immunization of animals with the DNA vector pVAX1-TSHR according to an experimentally selected scheme was effective for the formation of mouse splenocytes, secreting antibodies to TSHR, which were used for successful hybridization. This was confirmed by the results of determining antibody production to TSHR in murine blood sera. The level of antibody production remained high (titer more than 1:10.000) at the 8th week of the experiment. As a result of selection of individual clones according to the criteria of proliferative activity and stability of antibody production, the most stable cultures secreting mAbs against TSHR were selected.

Peptidomic profiling of endogenous peptides in the spleens of mouse models of Graves' disease

BackgroundGraves' disease (GD) is a common autoimmune thyroid disorder. The pathogenesis of GD involves an autoimmune response to the A subunit of the human thyrotropin receptor (hTSHR), although the specific mechanisms are not fully elucidated. MethodsThis study established a GD model by immunizing BALB/c mice with a recombinant adenovirus expressing the hTSHR A subunit. Spleen tissues were collected and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differentially expressed peptides (DEPs). Gene Ontology (GO) analysis and KEGG pathway analysis were further utilized to annotate the functions of these DEPs. Additionally, peptide bioactivity prediction and molecular docking studies were conducted using Alphafold and Pymol software, respectively, to assess the binding affinity of specific peptides to the hTSHR A subunit. ResultsThe GD mouse model was successfully established, and 1,428 DEPs were identified in the spleen, with 368 upregulated and 1,060 downregulated. Functional analysis indicated that these DEPs are mainly involved in cellular endocytosis, regulation of gene expression, and nucleocytoplasmic transport. Notably, molecular docking studies revealed that the abnormally highly expressed peptide HG2A-24aa demonstrated potential bioactivity and strong binding affinity with hTSHR-289aa. ConclusionThe specific bioactive peptides may play key roles in the pathogenesis of GD, particularly HG2A-24aa, which may have a significant role in the MHC II antigen presentation pathway mediated by the hTSHR A subunit.

PET Imaging of Differentiated Thyroid Cancer with TSHR-Targeted [89Zr]Zr-TR1402.

Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [89Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [89Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89Zr (t1/2 = 78.4 h, β+ = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [89Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [89Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T (P < 0.0001) and FTC133 (P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T (P < 0.0001) and TSHR+ FTC133 (P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [89Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference (P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference (P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [89Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

Abstract 54: Engineering chimeric antigen receptor (CAR) T cells using autoantigen binding domains for treatment of B cell malignancies with increased specificity

Abstract Introduction: Chronic inflammation often leads to cancer, and many autoimmune diseases lead to a significantly increased risk of developing cancer. Though many autoimmune diseases, like Graves’ Disease (GD), don’t have treatments that address this risk. Chimeric antigen receptor (CAR) T cells are an effective treatment for hematological cancers by eliminating all B cells, but a more targeted form of CAR T cells could eliminate specific B cell populations in B cell malignancies, including GD. Methods: To allow our CAR T cells to target specific B cell populations that cause GD, we engineered chimeric autoantigen receptors (CAAR) with epitopes of thyroid stimulating hormone receptor (TSHR) as the binding domain. This acts as bait for the anti-TSHR B cells. Jurkat T cells were lentivirally transduced with these CAAR plasmids. Anti-TSHR B cell lines were engineered by transducing Nalm6 cells with lentivirus for anti-TSHR B cell receptors. Cytotoxicity assays were performed on primary human TSHR CAAR T cells incubated with the anti-TSHR B cell lines. Cytokine production, proliferation, and cytotoxicity assays were performed on these coincubated cells to determine the efficacy of our TSHR CAAR T cells. Results: Two of the three Jurkat TSHR CAAR T cells showed significant binding to anti-TSHR antibodies (TRAbs). They also showed significant activation, measured by CD69 expression, when incubated with plate bound TRAbs, but not soluble TRAbs. The B cell lines were validated by binding to TSHR. The primary TSHR CAAR T cells showed significantly increased proliferation and production of TNF, IFN-y, and IL-2, when compared with controls. The TSHR CAAR T cells also had significant cytotoxic action against the anti-TSHR B cell lines, but not Nalm6 B cells. The cytotoxic function was comparable to our CD19 CAR T cell with the same activation domains, though it demonstrated specificity to only anti-TSHR B cells. Conclusion: TSHR CAAR T cells are a potentially curative treatment for GD that will stop chronic inflammation and could reduce GD patient’s risk of developing thyroid cancer. This is also an important development in CAR T cell therapies that allows for specific targeting of certain B cell populations. Citation Format: Abiagil Cheever, Hunter Lindsay, MacKenzie Hansen, Chloe Kang, K. Scott Weber, Kim O'Neill. Engineering chimeric antigen receptor (CAR) T cells using autoantigen binding domains for treatment of B cell malignancies with increased specificity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 54.

TSHR Variant Screening and Phenotype Analysis in 367 Chinese Patients With Congenital Hypothyroidism.

Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype-phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.

Graves' Orbitopathy Models: Valuable Tools for Exploring Pathogenesis and Treatment.

Graves' orbitopathy (GO) is the most common extrathyroidal complication of Graves' disease (GD) and severely affects quality of life. However, its pathogenesis is still poorly understood, and therapeutic options are limited. Animal models are important tools for preclinical research. The animals in some previous models only exhibited symptoms of hyperthyroidism without ocular lesions. With the improvements achieved in modeling methods, some progressive animal models have been established. Immunization of mice with A subunit of the human thyroid stimulating hormone receptor (TSHR) by either adenovirus or plasmid (with electroporation) is widely used and convincing. These models are successful to identify that the gut microbiota influences the occurrence and severity of GD and GO, and sex-related risk factors may be key contributors to the female bias in the occurrence of GO rather than sex itself. Some data provide insight that macrophages and CD8+ T cells may play an important pathogenic role in the early stage of GO. Our team also replicated the time window from GD onset to GO onset and identified a group of CD4+ cytotoxic T cells. In therapeutic exploration, TSHR derived peptides, fingolimod, and rapamycin offer new potential options. Further clinical trials are needed to investigate these drugs. With the increasing use of these animal models and more in-depth studies of the new findings, scientists will gain a clearer understanding of the pathogenesis of GO and identify more treatments for patients.

FRI533 A Novel Cre-loxP Based Mouse Model For Graves' Disease

Abstract Disclosure: D. Kim: None. Y. Bao: None. M. Kang: None. Y. Cho: None. Y. Kim: None. Y. Hwang: None. C. Ku: None. E. Lee: None. Objectives: Over years, various approaches have been used to model human Graves’ disease in mice, including transfected fibroblasts, and plasmid or adenoviral immunizations with the extracellular A subunit of the human thyrotropin receptor (hTSHRa). However, these models require a lot of effort for multiple dosing over several months and the results are very heterogeneous, limiting their use as a platform for the development of new therapeutics. Thus, we developed a novel Cre-loxP based mouse model for Graves' disease. Methods: To overcome the limitation of existing animal models, we made ESC-derived KI mouse expressing hTSHRa that will act as an antigen in a conditional manner by inserting the CAG-loxP-STOP-loxP-hTSHRa cassette into the mouse Rosa26 locus. TAT-Cre, a recombinant cell-permeant fusion cre-recombinase protein, was used for site-specific recombination to avoid leaky expression which may induce immune tolerance. Various concentrations of TAT-Cre were tested to avoid neutralization and produce adequate hyperthyroidism. Serum TSHR Ab and T4 levels were measured. Immunohistochemical examinations of the thyroid gland and orbits were performed. Results: Treatment with TAT-Cre within the range of 250-750U produced the most efficient TSHR Ab and significantly increased T4. Four weeks after TAT-Cre 500U administration, TSHR Ab levels were already significantly produced in all mice and T4 levels started to increase. At 8 and 12 weeks, TSHR Ab levels were produced at a very high levels in all mice and T4 levels were increased to a very high levels in over 80% of mice. After 3 months, MRI images did not show any significant protrusion. However, there was an enlargement in the volume of the orbital muscles. We also found thyroid enlargement and hypertrophy, orbital macrophage infiltration and fibrosis in Immunohistochemical examinations. Conclusions: A new mouse model for Graves' disease was successfully established by using the Cre-loxP system. After being optimized for orbitopathy induction, this model can be used as a platform for the development of novel therapeutics. Presentation: Friday, June 16, 2023

Establishment and Comparison of Two Different Animal Models of Graves' Orbitopathy.

The purpose of this study was to construct an animal model of Graves' ophthalmopathy (GO) by comparing recombinant adenovirus expressing human thyrotropin receptor A subunit (Ad-TSHR A) gene immunization and dendritic cell (DC) immunization. We evaluated the animal models that are closer to the pathology of human GO, and laid the foundation for the study of GO. Ad-TSHR A was injected intramuscularly into female BALB/c mice to induce the GO animal model. A GO animal model was constructed using TSHR combined with IFN-γ-modified primary DC immunized female BALB/c. The animal models constructed by the above two methods were evaluated in terms of ocular appearance, serology, pathology, and imaging to assess the modeling rate of the animal models, respectively. Both modeled mice exhibited increased serological indexes of free thyroxine (FT4) and TSH receptor antibodies (TRAbs) levels and decreased TSH (P < 0.01). Thyroid pathology analysis revealed the number of thyroid follicles increases, the size varies, and the follicular epithelial cells proliferate to varying degrees in a cuboidal or tall columnar pattern, with a small amount of lymphocytic infiltration visible. Adipose tissue behind the eyeball was accumulated, the muscle outside the eyeball was broken and fibrotic, and hyaluronic acid (HA) behind the eyeball was increased. The animal model of GO constructed by immunization of TSHR with IFN-γ-modified DC had a modeling rate of 60%, whereas that of Ad-TSHR A gene immunization was 72%. Both gene immunization and cellular immunization can be used to construct GO models, and the modeling rate of gene immunization is higher than that of cellular immunization. In this study, two innovative methods, cellular immunity and gene immunity, were used to establish GO animal models, which improved the success rate to a certain extent. To our knowledge, this study presents the first cellular immunity modeling idea of TSHR combined with IFN-γ for the GO animal model, which provides an animal model basis for understanding the pathogenesis of GO and developing new treatment methods.

Treatment with linsitinib, an IGF-1 receptor inhibitor, attenuates disease development and progression in an experimental model of Graves’ Orbitopathy

Abstract Study objective: The activity of linsitinib, a small molecule inhibitor of the insulin-like growth factor 1 receptor (IGF-1R), was assessed in a mouse model of Graves’ orbitopathy (GO). GO is the most common extra-thyroidal manifestation of Graves’ disease (GD), which is caused by autoantibodies against the thyroid stimulating hormone receptor (TSHR). Methods: GD was induced in mice by immunization with a human TSHR A-subunit encoding plasmid. Linsitinib was administered orally for four weeks either in parallel to or after disease induction. Endocrine orbitopathy and inflammation were determined by histology and MRI. Results: Immunization with the TSHR A-subunit induced hyperthyroidism, as well as CD3+ T-cell infiltration and macrophage infiltration into orbital muscle/adipose tissue. Linsitinib reduced these effects by up to 80% whether given prophylactically or after disease induction. In addition, linsitinib reduced the formation of brown adipose tissue in the orbit and displayed an additional effect on the immune system. An MRI using F19 imaging confirmed marked orbital inflammation, significant muscle edema and formation of brown fat in experimental GO, events that were abrogated upon application of linsitinib. Linsitinib’s pharmacologic effects were observed at a dose that did not impact the concentration of plasma glucose. Conclusion: Linsitinib improved GO-related endpoints in a TSHR autoantibody dependent mouse model of Graves’ disease. Both prophylactic and therapeutic intervention with linsitinib improved clinically relevant endpoints in the orbita and thyroid gland. Our results are consistent with the therapeutic hypothesis being evaluated in an ongoing Phase 2b clinical study (NCT05276063). This work was funded by Sling Therapeutics, Inc.

The Effect of Long-Term Inorganic Iodine on Intrathyroidal Iodothyronine Content and Gene Expression in Mice with Graves' Hyperthyroidism.

Background: The main molecular mechanism underlying acute suppression of iodine organification in normal thyroids after an excessive iodine load, that is, the Wolff-Chaikoff effect, is assumed to be suppression of iodine oxidation and iodothyronine synthesis. However, the mechanism underlying chronic antithyroid action of inorganic iodine in Graves' disease is not fully understood. Using a mouse model of Graves' hyperthyroidism, we examined changes in iodothyronine content and gene expression profiles in the thyroid glands after inorganic iodine loading. Materials and Methods: Graves' hyperthyroidism was induced and maintained in BALB/c mice by repeated immunizations of recombinant adenovirus expressing the human thyrotropin (TSH) receptor A-subunit. Hyperthyroid mice were left untreated (GD-C; n = 8) or treated with inorganic iodine for 12 weeks (GD-NaI; n = 8). We used unimmunized BALB/c mice as a control group (n = 10). In each mouse, serum thyroxine (T4) levels were measured with enzyme-linked immunosorbent assay (ELISA) at 4-week intervals. The intrathyroidal iodothyronine content and gene expression levels were, respectively, evaluated by mass spectrometry and RNA sequencing (RNA-seq) at the end of the experimental period. Results: Serum T4 levels in the GD-C group remained higher than in the control group, whereas those in the GD-NaI group declined to normal levels during the experimental period. Intrathyroidal triiodothyronine (T3), reverse T3 (rT3), and T4 contents in the GD-C group were higher than the control group, and rT3 and T4 were further increased in the GD-NaI group. The observed alterations in iodothyronine levels in the thyroid and sera may be explained by altered expression levels of genes for iodothyronine biosynthetic molecules, their transporter, and deiodinases. Conclusion: In this mouse model of hyperthyroidism, higher intrathyroidal accumulation of T4 and reduced gene expression data of iodothyronine transporters in the GD-NaI group suggest that chronic antithyroid action of iodine in Graves' disease involves suppression of hormone secretion.

Structure of full-length TSH receptor in complex with antibody K1-70™.

Determination of the full-length thyroid-stimulating hormone receptor (TSHR) structure by cryo-electron microscopy (cryo-EM) is described. The TSHR complexed with human monoclonal TSHR autoantibody K1-70™ (a powerful inhibitor of TSH action) was detergent solubilised, purified to homogeneity and analysed by cryo-EM. The structure (global resolution 3.3 Å) is a monomer with all three domains visible: leucine-rich domain (LRD), hinge region (HR) and transmembrane domain (TMD). The TSHR extracellular domain (ECD, composed of the LRD and HR) is positioned on top of the TMD extracellular surface. Extensive interactions between the TMD and ECD are observed in the structure, and their analysis provides an explanation of the effects of various TSHR mutations on TSHR constitutive activity and on ligand-induced activation. K1-70™ is seen to be well clear of the lipid bilayer. However, superimposition of M22™ (a human monoclonal TSHR autoantibody which is a powerful stimulator of the TSHR) on the cryo-EM structure shows that it would clash with the bilayer unless the TSHR HR rotates upwards as part of the M22™ binding process. This rotation could have an important role in TSHR stimulation by M22™ and as such provides an explanation as to why K1-70™ blocks the binding of TSH and M22™ without activating the receptor itself.

Expression and Purification of the Human Thyroid-Stimulating Hormone Receptor.

The thyroid-stimulating hormone receptor (TSHR) is a Class A G protein-coupled receptor (GPCR) that mediates signalling through the hypothalamic-pituitary-thyroid axis. Inappropriate activation of TSHR by autoantibodies or mutations, results in human disease such as Grave's disease and Hashimito's thyroiditis. Therefore, there is a need to develop novel therapeutics targeting the TSHR. Understanding the structure and mechanism of activation of this receptor would help elucidate the pathogenesis of disease and aid drug development. Here, we describe a method for the expression of the human TSHR in a mammalian cell line generated through a lentiviral expression system. The receptor is then purified by affinity chromatography in the ligand-free state and is suitable for structure determination by single-particle electron cryo-microscopy (cryo-EM).

A rapid homogenous bioassay for detection of thyroid-stimulating antibodies based on a luminescent cyclic AMP biosensor

Graves' disease (GD) is an autoimmune disease caused by antibodies to the thyroid stimulating hormone receptor (TSHR). The FDA-cleared Thyretain™ TSI bioassay is a highly specific method to detect thyroid stimulating antibodies (TSAb/TSI) in the blood of patients with autoimmune thyroid disease (AITD), particularly GD. To simplify the workflow of this bioassay and to support a semi-quantitative result, we have generated a stable CHO-K1 cell line expressing both a chimeric TSH receptor (TSHR-Mc4) and a luciferase-based homogeneous cAMP biosensor (GS luciferase). Here, we describe a rapid, real-time, homogenous bioassay (Turbo™ TSI Bioassay) to directly assess the functional activity of TSI and produce results in International Units of IU/L. The Turbo™ TSI bioassay works by measuring changes in the intracellular cAMP level induced by a G-protein coupled receptor (G-PCR) signaling cascade which is triggered by the binding of TSI to the TSHR. Upon binding to cAMP, the GS luciferase reporter is activated through conformational changes and generates light that can be measured in intact cells with a luminometer. The LoD and LoQ of the assay were determined to be 0.016 IU/L and 0.03 IU/L, respectively and the preliminary assay cutoff was determined to be 0.024 IU/L by ROC analysis using the Thyretain™ TSI bioassay results as reference. The analytical performance of the Turbo™ TSI bioassay is comparable to the Thyretain™ TSI bioassay as evidenced by similar EC50 values for a TSHR stimulating monoclonal antibody (M22). The specificity of the Turbo™ TSI bioassay was demonstrated by showing no response to a high concentration of a human monoclonal TSHR blocking antibody (K1–70). The precision of the assay was excellent with an overall within-laboratory precision <15% CV. When testing 198 clinical samples, the positive and negative percent agreement between the Turbo™ TSI and the Thyretain™ TSI bioassays were 98.7% and 93.5%, respectively. While both bioassays yield equivalent analytical and clinical performances, the Turbo™ TSI bioassay is much simpler to perform. It does not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time from about 21 h to 60 min. In addition, the same cell line showed its capability of detecting thyroid blocking antibodies (TBAb/TBI) in a competitive format. The Turbo™ TSI bioassay is user-friendly and is a very promising advancement to aid the diagnosis of autoimmune thyroid disease (AITD).

Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves' Orbitopathy.

Background. To obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1. Methods. The effects of 100 nM OMD or 10 μM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs). Results. The physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs. Conclusions. The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

A Promising Mouse Model of Graves' Orbitopathy Induced by Adenovirus Expressing Thyrotropin Receptor A Subunit.

Background: Graves' orbitopathy (GO) is the most common and serious manifestation of Graves' disease (GD). It is characterized by orbital inflammation and tissue remodeling. Although several GO models have been reported, most lack a full assessment or mechanistic evaluation. Here, we established a promising mouse model mimicking many aspects of human GO with a frequency of 70% and characterized the key role of T cells in the progression of GO. Methods: An adenovirus expressing the human thyrotropin (TSH) receptor A subunit (Ad-TSHRA) was injected in the muscles of female BALB/C mice nine times to induce GO. At predetermined time points, histological examinations of retrobulbar tissues and thyroid glands were performed to dynamically monitor changes; serum autoantibodies and total thyroxine levels were examined to evaluate thyroid function. Flow cytometry of CD4+ T cell subgroups and RNA sequencing (RNA-Seq) of splenocytes were also performed to explore the underlying mechanism. Results: After nine injections, 7 of 10 mice challenged with Ad-TSHRA developed the orbital changes associated with GO. Seven mice manifested retrobulbar fibrosis, and four mice showed adipogenesis. Exophthalmia, conjunctival redness, and orbital lymphocyte infiltration were also observed in a subset of mice. The orbitopathy was first detected after seven injections and followed the hyperplastic change observed in thyroids after four injections. Flow cytometry revealed increased proportions of Th1 cells and decreased proportions of Th2 cells and regulatory T (Treg) cells in the splenocytes of GO mice. This change in CD4+ T cell subgroups was confirmed by orbital immunohistochemical staining. Genes involved in T cell receptor signaling, proliferation, adhesion, inflammation, and cytotoxicity were upregulated in GO mice according to the RNA-Seq; a trend of upregulation of these GO-specific genes was observed in mice with hyperthyroidism without orbitopathy after four injections. Conclusions: A GO mouse model was successfully established by administering nine injections of Ad-TSHRA. The model was achieved with a frequency of 70% and revealed the importance of T cell immunity. A potential time window from Graves' hyperthyroidism to GO was presented for the first time. Therefore, this model could be used to study the pathogenesis and novel treatments for GO.

Thyroid-stimulating hormone receptor (TSHR) fusion proteins in Graves' disease.

Graves' disease is an autoimmune disorder, which is characterized by stimulatory antibodies targeting the human thyrotropin receptor (TSHR), resulting in hyperthyroidism and multiple organ damage. We systematically investigated monomeric and dimeric fusion proteins of the A subunit of TSHR for efficacy to bind to the monoclonal patient antibody M22, to interact with Graves' patient serum samples, and to impact on anti-TSHR antibody titers, hyperthyroidism, tachycardia and other in vivo read-outs in a long-term mouse model of Graves' disease induced by immunization with a recombinant adenovirus encoding TSHR A. Binding assays and functional measurements of TSHR-dependent cAMP formation showed binding of monomeric TSHR-His and dimeric TSHR-Fc to the anti-TSHR antibody M22 at low-effective concentrations (EC50 of 5.7 nmol/L and 8.6 nmol/L) and inhibition of the effects of this antibody at high efficiencies (IC50 values of 16-20 nmol/L). Both proteins also block the effects of polyclonal anti-TSHR antibodies occurring in Graves' patient sera with somewhat lower average efficiencies (mean IC50 values of 29 nmol/L and 68 nmol/L). However, in vivo characterization of epicutaneous patch administrations of TSHR-Fc at doses of 0.3 and 0.6 mg/kg body weight in a murine Graves' disease model did not result in any improvement of disease parameters. In conclusion, high affinity binding of TSHR-Fc to pathological anti-TSHR antibodies was not matched by efficacy to improve Graves' disease parameter in a long-term mouse model.

A Novel Long-Term Graves’ Disease Animal Model Confirmed by Functional Thyrotropin Receptor Antibodies

Introduction: A novel long-term murine model for Graves’ disease (GD) using repeated, long-term immunizations with recombinant adenovirus expressing the extracellular A-subunit of the human thyrotropin receptor (Ad-TSHR) was applied to evaluate the functional anti-TSHR-antibody (TSHR-Ab) profile. Methods: BALB/c mice received 7 immunizations with either 10¹⁰ plaque-forming units of Ad-TSHR or control Ad-GFP. Naïve (nonimmuized native) mice were also studied. Three 3-weekly immunizations were followed by 4-weekly boosts until the 7th immunization. Blocking (TBAb) and stimulating (TSAb) TSHR-Ab were measured with bioassays. Assay cut-offs for TBAb/TSAb were at 34% inhibition and a specimen-to-reference ratio (SRR) of 140%. Results: Nineteen (8 Ad-TSHR-, 4 Ad-GFP-immunized, and 7 native) mice were investigated. All native mice were negative for TSHR-binding inhibitory immunoglobulins (TBII) prior to immunization. Native and Ad-GFP mice were negative in weeks 17 and 27 for TBII and TBAb/TSAb. In native mice, the free thyroxine (fT4) levels (median [25th percentile; 75th percentile]) were in the upper normal range (1.2 ng/mL [1.1; 1.6]) prior to immunization, at weeks 17 (2.2 ng/mL [2.1; 2.4]) and 27 (1.4 ng/mL [1.1; 1.7]), respectively. In contrast, in Ad-TSHR-immunized mice, fT4 values were markedly increased at weeks 17 (4.4 ng/mL [3.9; 6]) and 27 (4.5 ng/mL [4.2; 6]) compared to those in Ad-GFP mice (2 ng/mL [1.8; 2.1] and 1.4 ng/mL [1.1; 1.6]), respectively (p = 0.0008, p = 0.001). In contrast, at week 17, in Ad-TSHR mice, the mean TBII, TBAb, and TSAb levels were 40 IU/L (40; 40); 62% inhibition (38; 69), and 116% SRR (97; 185), respectively; at week 27, they were 40 IU/L (39; 40); 65% inhibition (34; 80) and 95% SRR (63; 187), respectively. Three serum samples from Ad-TSHR mice (38%) demonstrated dual TBAb/TSAb positivity. Conclusions: TBAb/TSAb were highly prevalent in Ad-TSHR-immunized mice, thus confirming the successful establishment of a novel, long-term murine model for GD. All TBAb- and TSAb-positive Ad-TSHR-immunized mice were TBII-positive. Thus, the binding immunoassay did not differentiate between TSHR-Ab functionality.

β-Arrestin 1 in Thyrotropin Receptor Signaling in Bone: Studies in Osteoblast-Like Cells.

A direct action of thyrotropin (TSH, thyroid-stimulating hormone) on bone precursors in humans is controversial. Studies in rodent models have provided conflicting findings. We used cells derived from a moderately differentiated osteosarcoma stably overexpressing human TSH receptors (TSHRs) as a model of osteoblast precursors (U2OS-TSHR cells) to investigate TSHR-mediated effects in bone differentiation in human cells. We review our findings that (1) TSHR couples to several different G proteins to induce upregulation of genes associated with osteoblast activity—interleukin 11 (IL-11), osteopontin (OPN), and alkaline phosphatase (ALPL) and that the kinetics of the induction and the G protein-mediated signaling pathways involved were different for these genes; (2) TSH can stimulate β-arrestin-mediated signal transduction and that β-arrestin 1 in part mediates TSH-induced pre-osteoblast differentiation; and (3) TSHR/insulin-like growth factor 1 (IGF1) receptor (IGF1R) synergistically increased OPN secretion by TSH and IGF1 and that this crosstalk was mediated by physical association of these receptors in a signaling complex that uses β-arrestin 1 as a scaffold. These findings were complemented using a novel β-arrestin 1-biased agonist of TSHR. We conclude that TSHR can signal via several transduction pathways leading to differentiation of this model system of human pre-osteoblast cells and, therefore, that TSH can directly regulate these bone cells.

Practical applications of studies on the TSH receptor and TSH receptor autoantibodies

Studies on the TSH receptor (TSHR) have numerous practical applications in vitro and in vivo. For example human monoclonal autoantibodies (MAbs) to the TSHR are useful reagents for in vitro diagnostics. Measurement of TSHR autoantibodies (TRAbs) is helpful in diagnosis and management of autoimmune thyroid disease. Currently available highly sensitive and specific assays to measure TRAbs use the human TSHR MAb M22 instead of the TSH. Furthermore, preparations of the human TSHR MAb M22 are useful as the World Health Organisation International Standard for thyroid stimulating antibody and for calibration of the assays for measuring TRAbs. Preparations of thermostabilised TSHR extracellular domain have recently become available and this is likely to have an impact on improvements in specificity testing for TRAb assays. In addition the stable TSHR preparations have practical application for specific immunoadsorption of patient serum TRAbs. Human TSHR MAbs also have promising prospects as new therapeutics. Autoantibodies with TSHR antagonistic activities are “natural” inhibitors of TSHR stimulation and are expected to be helpful in controlling TSHR activity in patients with Graves’ disease, Graves’ ophthalmopathy and thyroid cancer.

TSH Receptor Homodimerization in Regulation of cAMP Production in Human Thyrocytes in vitro.

Thyrotropin hormone (TSH) was reported to exhibit biphasic regulation of cAMP production in human thyroid slices; specifically, upregulation at low TSH doses transitioning to inhibition at high doses. We observed this phenomenon in HEK293 cells overexpressing TSH receptors (TSHRs) but in only 25% of human thyrocytes (hThyros) in vitro. Because TSHR expression in hThyros in vitro was low, we tested the hypothesis that high, in situ levels of TSHRs were needed for biphasic cAMP regulation. We increased expression of TSHRs by infecting hThyros with adenoviruses expressing human TSHR (AdhTSHR), measured TSH-stimulated cAMP production and TSHR homodimerization. TSHR mRNA levels in hThyros in vitro were 100-fold lower than in human thyroid tissue. AdhTSHR infection increased TSHR mRNA expression to levels found in thyroid tissue and flow cytometry showed that cell-surface TSHRs increased more than 15-fold. Most uninfected hThyro preparations exhibited monotonic cAMP production. In contrast, most hThyro preparations infected with AdhTSHR expressing TSHR at in vivo levels exhibited biphasic TSH dose responses. Treatment of AdhTSHR-infected hThyros with pertussis toxin resulted in monotonic dose response curves demonstrating that lower levels of cAMP production at high TSH doses were mediated by Gi/Go proteins. Proximity ligation assays confirmed that AdhTSHR infection markedly increased the number of TSHR homodimers. We conclude that in situ levels of TSHRs as homodimers are needed for hThyros to exhibit biphasic TSH regulation of cAMP production.