MON-178 Vitamin D Treatment of Patients with Type 1 Diabetes Reduces RORy, FoxP3 and CD4 Gene Expression in T Helper Cells

Background : Vitamin D (VD) deficiency and the dysregulation of T helper (Th) cells contribute to the pathogenesis of type 1 diabetes (T1D). Based on the modulatory effects of VD on immune cells, we investigated the gene expression of transcription factors (TFs) regulating the Th cell differentiation before and after VD treatment in a pilot trial. Materials and methods : Nineteen patients with T1D (11 females and 8 males) were randomized and treated for three months with 4000 IU/day cholecalciferol. The gene expression of four Th cell specific TFs (Th1/T-bet, Th2/GATA3, Th1/RORy and Treg/FoxP3) as well as the Th cell surface molecule CD4 was measured before (V1) and after three months (V3) of VD treatment. Th cells were isolated by density gradient centrifugation from venous blood samples and subsequently enriched by magnetic sorting with antibodies. Total RNA from the selected cells was extracted, transcribed and analyzed by RT-qPCR. Transcription levels were calculated using the comparative cycle threshold (CT) method (values presented as 2 -[CTtarget -CT18S] = 2 -[∆CT] ). VD-therapy effects on the gene expression were evaluated by the differences between V1 and V3 using the Wilcoxon matched-pairs test. The median differences are given with Hodges-Lehmann estimator (HLS) and 95% confidence interval (CI). Results: The 25(OH)D 3 levels increased to 39.1 ng/ml (HLS: 17.22 ng/ml; 95% CI 9.3 to 23.9, p=8 x10 -4 ) after three months cholecalciferol treatment. In response to this the gene expression of the two TFs FoxP3 (HLS: -12.68; 95% CI -21.47 to -3.36, p=0.01) and RORy (HLS: -1.85; 95% CI -4.68 to 0.02,p trend =0.07) were downregulated. Additionally, a reduced gene expression of CD4 (HLS: -128; 95% CI -452.36 to -15.46, p=0.009) was detected. Notably, all these gene expression changes occurred in women (FoxP3 HLS: -17.30, p=0.02; RORy HLS: -3.96, p=0.01 and CD4 HLS: -146, p=0.007) but not in men. Furthermore, the downregulation of FoxP3 gene expression after VD supplementation was found in patients who had a 25(OH)D 3 level below 20 ng/ml (n=9) at study entry (HLS: -17.36;p=0.03). Changes in the gene expressions of T-bet and GATA3 were not significant. Conclusion: Elevated 25(OH)D 3 levels after high dose VD therapy result in downregulated genes (FoxP3, RORy and CD4) which are involved in the differentiation of effector Th cells (Treg and Th17). These VD effects occur predominantly in female patients, highlighting a gender specificity of the Th cell respon