measurement-of-t-cell-alloreactivity-using-imaging-flow-cytometry

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

University Health Network

Graves' orbitopathy (GO) is a complication in Graves' disease (GD) but mechanistic insights into pathogenesis remain unresolved, hampered by lack of animal model. The TSH receptor (TSHR) and perhaps IGF-1 receptor (IGF-1R) are considered relevant antigens. We show that genetic immunization of human TSHR (hTSHR) A-subunit plasmid leads to extensive remodeling of orbital tissue, recapitulating GO. Female BALB/c mice immunized with hTSHR A-subunit or control plasmids by in vivo muscle electroporation were evaluated for orbital remodeling by histopathology and magnetic resonance imaging (MRI). Antibodies to TSHR and IGF-1R were present in animals challenged with hTSHR A-subunit plasmid, with predominantly TSH blocking antibodies and were profoundly hypothyroid. Orbital pathology was characterized by interstitial inflammation of extraocular muscles with CD3+ T cells, F4/80+ macrophages, and mast cells, accompanied by glycosaminoglycan deposition with resultant separation of individual muscle fibers. Some animals showed heterogeneity in orbital pathology with 1) large infiltrate surrounding the optic nerve or 2) extensive adipogenesis with expansion of retrobulbar adipose tissue. A striking finding that underpins the new model were the in vivo MRI scans of mouse orbital region that provided clear and quantifiable evidence of orbital muscle hypertrophy with protrusion (proptosis) of the eye. Additionally, eyelid manifestations of chemosis, including dilated and congested orbital blood vessels, were visually apparent. Immunization with control plasmids failed to show any orbital pathology. Overall, these findings support TSHR as the pathogenic antigen in GO. Development of a new preclinical model will facilitate molecular investigations on GO and evaluation of new therapeutic interventions.

Cutting edge: retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves' orbitopathy induced by thyrotropin receptor plasmid-in vivo electroporation.

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.

Preclinical models of Graves' disease and associated secondary complications.

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.

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.

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.

Article	Year
Cutting edge: retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves' orbitopathy induced by thyrotropin receptor plasmid-in vivo ... Endocrinology\| PubMed ID: 23900776	2013
Preclinical models of Graves' disease and associated secondary complications. Current pharmaceutical design\| PubMed ID: 25777757	2015
Comparative Assessment of Female Mouse Model of Graves' Orbitopathy Under Different Environments, Accompanied by Proinflammatory Cytokine and T-Cell Responses to Thyrotropin Hormone Receptor... Endocrinology\| PubMed ID: 26872090	2016
Pathogenic Phenotype of Adipogenesis and Hyaluronan in Orbital Fibroblasts From Female Graves' Orbitopathy Mouse Model. Endocrinology\| PubMed ID: 27552248	2016

Sajad Moshkelgosha

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