Vitamin D ameliorates NETosis and Th17/Treg imbalance in experimental autoimmune thyroiditis

BACKGROUND: Neutrophil extracellular traps (NETs) have been implicated in various autoimmune diseases; however, their role in Hashimoto's thyroiditis (HT) remains poorly understood. This study aimed to characterize NETs formation, explore its association with thyroid dysfunction and adaptive immunity, and evaluate the therapeutic potential of vitamin D (VD) in experimental autoimmune thyroiditis (EAT). METHODS: EAT was induced in BALB/c mice via thyroglobulin immunization combined with excess iodine intake. The VD group received additional intraperitoneal calcitriol supplementation. Thyroid histopathology, MHC-II expression, thyroid antibodies (TGAb and TPOAb), plasma DNase-I and 1,25(OH)₂D₃ levels, NETs formation, and splenic T cell subsets (Th17, Treg, Th1, Th2) and cytokines were assessed. Parallel experiments were conducted using neutrophils isolated from HT patients. RESULTS: Neutrophils from both EAT mice and HT patients exhibited enhanced NETosis compared to controls. EAT mice showed lower levels of DNase-I, 1,25(OH)₂D₃, Treg, and Th1 cells, along with higher Th17 and Th2 cells, elevated Th17/Treg ratio, and heightened thyroid MHC-II expression. NETs levels positively correlated with Th17, Th2, and MHC-II expression, and negatively with Treg, Th1, 1,25(OH)₂D₃, and DNase-I. VD supplementation mitigated thyroiditis and TPOAb levels, suppressed NETs formation, and reduced the Th17/Treg ratio and IL-17 levels. CONCLUSIONS: NETs contribute to Th17 bias and MHC-II over-expression in HT, suggesting a role in shaping the adaptive immune response. Vitamin D restrains NETosis and restores T-cell homeostasis, highlighting its potential as an adjunctive therapy for HT.