Thyroglobulin epitopes and peptide repertoire in autoimmune thyroiditis: role of iodine intake and IFNα

Introduction: Thyroglobulin (Tg) is a key antigen for the development of Autoimmune Thyroid Disease (AITD). An increase in dietary iodine uptake from insufficient to sufficient values induces an increase of thyroid autoimmunity and the unmasking of the B epitope, recognized by human TgAb, on Tg. No data about the effect of an excessive iodine intake are available. IFNα, a cytokine released during viral infections, is a significant trigger of AITD. IFNα enhances the transcription of Tg gene and simultaneously accelerates the lysosomal degradation of the Tg protein. In vitro studies indicate that Tg cleavage by cathepsin L (CTS-L), a lysosomal protease, results in the formation of Tg.2098, a prominent T-cell epitope associated with AITD. Objectives: To compare the expression of TgAb epitopes in sera of Hashimoto’s thyroiditis (HT) patients from areas with adequate, slightly and markedly excessive iodine intake. To test the hypothesis that IFNα modifies the immunogenic peptide repertoire of Tg by CTS-L. Methods: Inhibition of Tg-TgAb binding was evaluated in HT patients from Italy (Ita, n. 12), Nagasaki (Nag, n. 11) and Sapporo (Sap, n. 11). Median urinary iodine in these cohorts were 124, 363 and 1015 μg/L. Tg-TgAb binding was inhibited by 4 recombinant human Tg-Ab Fabs that recognize Tg epitopes A, B, C and D. We exposed ML-1 thyroid cells to IFNα and evaluated the expression of CTS-L through western blotting. We performed knockdown of CTS-L using siRNA to analyze Tg expression in ML-1 cells treated with IFNα. The role of CTS-L in the generation of Tg.2098 was assessed in ML-1 cells treated with both IFNα and the CTS-L inhibitor (KPG94) via immunofluorescence (IF). To investigate the mechanisms by which IFNα regulates CTS-L in thyroid cells, we utilized pathway expression in IFNα-treated thyrocytes. Results: Levels of inhibition were similar for TgAb-Fab A (Ita: 47 ± 9%; Nag: 41 ± 11%; Sap: 32 ± 10%, p = 0.095) and TgAb-Fab B (Ita: 31 ± 11%; Nag: 36 ± 11%; Sap 37± 7%; p = 0.561), different for TgAb-Fab C (Ita 26 ± 5%; Nag: 40± 12%; Sap 31 ± 7%; p = 0.015) and TgAb-Fab D (Ita: 24 ± 7%; Nag 34 ± 8%; Sap 31 ± 10%; p = 0.025). In pairwise comparison, the level of inhibition was lower in Ita sera for TgAb-Fab C (p = 0.009 vs Nag) (p = 0.037 vs Sap) and TgAb-Fab D (p = 0.027 vs Nag) (p=0.017 vs Sap). Exposure to IFNα resulted in increased CTS-L expression in ML-1 cells. Knockdown of CTS-L led to a reduction, whereas overexpression resulted in an increase of IFNα-induced Tg degradation. Pathway analyses revealed that IFNα activates p38MAPKand inhibition of p38MAPK decreased CTSL levels induced by IFNα. IF studies showed elevated levels of Tg.2098 in ML-1 cells treated with IFNα, while treatment with CTS-L inhibitor reduced IFNα-induced Tg.2098 levels. Conclusions: We have previously shown that a rise in urinary iodine from 86.5 to 112.5 μg/L induced the unmasking of epitope B on Tg. A further increase (up to 363 μg/L) induces a higher expression of epitopes C and D. An additional increase (up to 1015 μg/L) does not lead to a different recognition of the 4 epitopes. IFNα enhances CTSL expression in the thyroid by p38MAPK, leading to the degradation of Tg into immunogenic peptides like Tg.2098. This finding introduces a novel mechanism for the virally triggered onset of AITD in susceptible populations.