Endocrine Abstracts

JOINT2912

Hashimoto’s thyroiditis (HT) is a common autoimmune disorder of the thyroid gland. The appearance of anti-thyroid antibodies (ATAs) against thyroid peroxidase (TPO) and thyroglobulin (TG), and the infiltration of the thyroid gland by mononuclear cells are characteristic markers of HT. Although it is not obligatory, HT can lead to hypothyroidism at an advanced stage of the disease (indicated by the increased level of thyroid-stimulating hormone, TSH), against which levothyroxine replacement therapy has been proved to be beneficial. Since HT is considered to be a T cell-mediated disease primarily, we designed a transcriptome analysis experiment (RNA-seq) to examine gene expression alterations in T lymphocytes in HT and the effect of levothyroxine treatment. Peripheral blood samples were collected from ATA+ hypothyroid HT patients (TPO and TG antibody positivity, abnormal elevated TSH level) before and after levothyroxine replacement therapy and from the age-matched healthy controls. T cells were isolated from the blood by magnetic separation and then total RNA was purified. Whole transcriptome sequencing (bulk) was performed using the Illumina NovaSeq 6000 platform, with QuantSeq 3’ mRNA library preparation and single-end sequencing with 75bp read length, in order to screen for differentially expressed (DE) genes. Following the identification of DE genes, we carried out a functional enrichment analysis to investigate which biological processes and signaling pathways are affected by these changes. Our screen revealed that the vast majority of DE genes were up-regulated in T lymphocytes in HT, approximately 85 percent, compared to the healthy control. Interestingly, the contrary could be observed in the same disease patients after levothyroxine treatment, where 262 of the 308 DE genes were down-regulated in T cells. Between the control and the treated groups, DE genes were present in a notably lesser amount. By examining changes between the healthy vs Hashimoto and the Hashimoto vs treated comparisons, we found 181 identical genes that exhibited a clear switch in their expression pattern from an up-regulated to a down-regulated state. Several upstream regulators, such as interleukin-1A (IL-1A), IL-1B and tumor necrosis factor (TNF) were activated in HT and became inhibited following levothyroxine addition. In addition, we observed a similar activation-inactivation phenomenon in many signaling pathways (IL-4 and IL-13, IL-6, IL-17, TNF, nuclear factor-kappa B and transforming growth factor beta signaling), suggesting that altered immunological processes characteristic of the disease may be reversed by applying levothyroxine therapy.