Mixed TH1 and TH17 cell responses in murine granulomatous experimental autoimmune thyroiditis induced by hydrophobic intermediates of thyroglobulin dissociation/unfolding in urea

Background: A granulomatous experimental autoimmune thyroiditis (G-EAT), with follicle lysis, hystiocytic/granulocytic infiltration and fibrosis, was induced in CBA/J(H-2k) mice by immunization with hydrophobic intermediates of the dissociation/unfolding of human thyroglobulin (hTg) in urea, namely, partially unfolded hTg monomers (PUM) and non-natively reassociated dimers (NND). Methods: Female CBA/J(H-2k) mice were immunized with native hTg, partially unfolded monomers (PUM) or non-native dimers (NND), obtained by denaturation of hTg in 3.5 M urea at pH 9.0, and separated by sucrose density centrifugation. On day 18, we evaluated thyroid histology, splenocyte proliferation and multiple cytokine levels in the supernatants, by fluorescent bead flow cytometry. Results: Proliferative responses of splenocytes from mice immunized with PUM and NND to the latter antigens in vitro were stronger than those of mice immunized and restimulated with native hTg. They were marked by the production of higher levels of IFN- γ, TNF-α, IL-2, IL-6 and IL-10, compared with native hTg, and by a unique IL-17 response. Significant cytokine production occurred also with control splenocytes exposed to PUM and NND, which points to the participation of APCs and NK cells in the effects of unfolded hTg. Conclusions: The greater ability of partially unfolded hTg species with increased hydrophobicity to induce autoimmune thyroid damage, with respect to native hTg, seemingly relied upon their ability to induce a stronger TH1 and a unique TH17 cell response. Further investigation is warranted regarding human AITD forms possibly triggered by hTg folding/dimerization defects, or by the release of unfolded, nascent hTg molecules upon thyroid cell damage.