Analyzing the role of thyroidal CD40 expression in the pathogenesis of Graves' disease

Graves' disease (GD) is characterized by the production of high levels of thyroid stimulating hormone receptor (TSHR) auto-antibodies which activate the TSHR and cause goiter and thyrotoxicosis. A C/T single nucleotide polymorphism (SNP) in the CD40 gene at the -1 position of the 5'UTR, in the Kozak sequence, was shown to be associated with GD, increasing the risk for GD by 30-80%. Functionally, the C allele of this SNP has been shown to drive increased CD40 gene translation, and cellular CD40 protein expression. In one study, we showed a statistical association of the CC genotype in GD with high titers of thyroid specific antibodies, more precisely, pathogenic, disease causing antibodies.;In another study, we analyzed a known CD40 copy number variant (CNV) for association with Graves' disease, since duplications in the CD40 gene could also result in an increased protein expression of CD40. We found no duplications or deletions in the locus of a known CD40 CNV, suggesting that CNVs in this region are rare, and that the entire genetic risk seen with CD40 in GD is likely to be attributed to non-CNV genomic variations, mostly the CD40 5'UTR Kozak sequence SNP we have identified.;Based on these findings, my hypothesis is that thyroidal CD40, through tissue specific mechanisms, is involved in the pathogenesis of Graves' disease, specifically in the process of pathogenic antibody production. To test this, I created transgenic (TG) mice that over-express CD40 in the thyroid, and then induced the currently accepted experimental autoimmune Graves' disease (EAGD) model, first developed by Nagayama. I demonstrate that thyroid specific expression is not necessary for onset of EAGD, it is important in augmenting the production of thyroid specific antibodies, as there was a significant increase in THSR antibodies (TRAb) in TG mice compared to WT. Moreover, these antibodies were stimulatory in nature, as there was also a significant increase in the serum thyroid hormone levels in TG mice compared to WT. This suggests that tissue specific CD40 mediates stimulatory, pathogenic antibody production in GD. In addition, when EAGD was induced in mice that lacked CD40 expression in non-bone marrow derived cells, including thyrocytes, fewer mice developed thyroid specific antibodies, levels of these antibodies were decreased in the mice that did develop antibodies, and in the percentage of mice developing EAGD was decreased. In total, these observations support the hypothesis that CD40 augments the production of pathogenic, tissue specific thyroidal antibodies in GD. Mechanistically, cytokine analysis demonstrated that transgenic mice had significantly higher levels of IL-6, and CD40 activation in human primary thyroid cells also resulted in secretion of IL-6. Additionally, blocking IL-6 action in EAGD mice resulted in decreased levels of TRAb in both WT and TG mice, although only this decrease was significant in TG mice, but not in the WT mice. Therefore, CD40 stimulation during times of local inflammation (i.e. by excess iodine or infection) could trigger a bystander mechanism through thyroidal cytokine release and recruitment of immune cells to the target tissue, resulting in tissue specific antibody production. This, when other predisposing factors are present, can lead to the onset of GD.