Molecular Mechanisms of Graves' Ophthalmopathy. A focus on smoking and radioiodine

Abstract: Graves’ disease (GD) is an autoimmune disease characterized by hyperthyroidism and is caused by an interplayof genetic and environmental factors. One-third of patients with GD develop Graves’ ophthalmopathy (GO). Keyprocesses in the pathogenesis of GO are inflammation and adipogenesis in orbital tissue. Strong risk factorsassociated with the development of GO are smoking and radioiodine treatment for hyperthyroidism. The aim ofthis thesis was to explore the molecular mechanisms behind the effects of smoking and radioiodine treatment onthe pathogenesis of GO.In Study I, microarray results showed upregulation of IL-1ß, IL-6 and adipocyte-related immediate early genes(IEGs), CYR61 and PTGS2, in intraorbital adipose/connective tissue from smokers compared with nonsmokerswith active GO. We confirmed the results with Reverse transcription polymerase chain reaction (RT-PCR). Usingbioinformatic tools, we investigated whether the overexpressed genes were associated with pathways upregulatedin adipogenesis. We found the overexpressed genes to be significantly associated with pathways involvinginflammation, adipogenesis, and immune responses.In Study II, we studied the direct effect of cigarette smoke extract (CSE) on the gene expression of IL-1ß, IL-6,CYR61, and PTGS2 and on the adipogenesis process using an in vitro model. Orbital fibroblasts (OFs) wereisolated from intraorbital adipose/connective tissue from GO patients. The OFs were exposed to CSE, and geneexpression was measured. We found that CSE alone could upregulate the gene expression of IEGs, IL-1ß, and IL-6 but not the late adipogenic genes SCD and PPARγ. Moreover, we investigated whether simvastatin coulddownregulate IEGs, IL-6 and adipogenesis. Simvastatin downregulated IEGs, IL-6, and adipogenesis in OFs.Additionally, we found that IGF-1 treatment in 3T3-L1 preadipocytes led to mature adipocytes.In Study III, we investigated the effect of CSE exposure and simvastatin and diclofenac treatment on peripheralblood mononuclear cells (PBMCs) isolated from newly diagnosed GD patients. We found that CSE exposurealone could upregulate the expression of PTGS2, IL-1ß, and IL-6 and the release of PGE2, IL-1ß, and IL-6.Furthermore, the proliferation of B lymphocytes and T lymphocytes was upregulated in response to CSEexposure. Simvastatin and diclofenac downregulated the gene expression of PTGS2, IL-1ß, and IL-6 and therelease of PGE2, IL-1ß, and IL-6 by PBMCs. Furthermore, the proliferation of B- and T lymphocytes wasdownregulated. The effects of simvastatin and diclofenac were even stronger when the treatments werecombined. Additionally, IGF-1-treated PBMCs upregulated the proliferation of B- and T lymphocytes.In Study IV, GD patients were treated with radioiodine (RI). Thyrotropin receptor autoantibody (TRAb) levels weremeasured 3 months after treatment. DNA was collected using buccal swabs. We found that TRAb levels weresignificantly increased in 70% percent of the patients 3 months after RI treatment. TRAb levels were significantlyelevated in the proportion of patients who developed GO after RI treatment compared to those GD patients that didnot develop GO after RI treatment. A single nucleotide polymorphism (SNP) rs231775 in the CTLA-4 gene wasassociated with TRAb levels in GD patients after RI treatment.