Endocrine Abstracts

Introduction: Autoimmune Thyroid Diseases (AITD) are one of the most prevalent autoimmune diseases in industrialized countries (5% of population). The two main phenotypes of AITD, Hashimoto thyroiditis (HT) and Graves’ disease (GD), are both characterized by the presence of circulating thyroid antibodies and infiltration by autoreactive lymphocytes in the thyroid gland and sometimes the orbit. One of the most studied mechanisms underlying AITD is the imbalance between immune activation and immune homeostasis of CD4+CD25- cells or T effector cells (Teff) and CD4+CD25+FOXP3+ regulatory T cells (Treg). Histone deacetylases (HDACs) are enzymes that exert postranslational modifications at protein level. HDAC9 interacts with FOXP3, the master regulator of Tregs, leading to an imbalance in Treg function. We have recently reported an increase expression of HDAC9 in Treg cells from AITD patients.

Objective: To investigate the in vitro effects of HDAC inhibitors (trichostatin A (TsA) a pan-inhibitor, TMP-269 a class IIa inhibitor and the FDA approved pan-inhibitor, suberanilohydroxamic acid (SAHA/Vorinostat)) on human freshly isolated CD4+CD25- T effector cells from AITD patients.

Methods: Toxicity assays were evaluated using LIVE/DEAD Viability-Cytotoxicity Kit on T cell proliferation by each inhibitor. Treg suppression assays were carried out in healthy controls and AITD patients. CD4+CD25+ Tregs were isolated from fresh PBMC using CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec). To evaluate proliferation Teff cells were CFSE-labeled, and added to wells in serial dilutions giving Treg/Teff ratios of 0:1, 1:1, 1:2; 1:4 and 1:8 and in the presence or absence of differing concentrations of HDACi and using DMSO as control.

Results: Toxicity assays revealed us that TMP269 and SAHA demonstrate the same number and viability as control cells. On the contrary, TsA decreased significantly the viability at the minimal concentration used, discarding this inhibitor from our assays. Suppression assays using the TMP269 inhibitor did not showed significantly effects on the proliferation of CD25- T cells. However, SAHA caused a mild to moderate impairment of CD25- division.

Conclusions: Among all the inhibitors assessed, SAHA did not exert a toxic effect in cells and had a significantly decrease on Teff proliferation compared to TMP269. Our study also showed that the impaired proliferation of CD4+CD25-Teff cells by SAHA, was not only by a specific Treg mediated effect, but also by the decrease in the CD4+CD25- cell division rate. These findings suggest that HDAC inhibition by SAHA may serve as a possible treatment of inflammation in AITD.