Hashimoto’s thyroiditis (HT) is the most common thyroid disease, and cause of hypothyroidism with an incidence of 0.3−1.5 per 1,000 individuals and is steadily increasing (1). HT shows a relatively low prevalence in Asian countries, with an estimated prevalence of around 5.8%, and demonstrates a clear female predominance with a female-to-male ratio of approximately 4:1 (2). HT is characterized by the presence of autoantibodies, including thyroglobulin (TG), thyroid peroxidase (TPO), and the thyrotropin receptor (TSHR), indicating an autoimmune response to self-antigens, leading to chronic inflammation and disruption of thyroid function (3). The risk factors for HT involve a complex interplay between endogenous and exogenous factors (4). Recent studies have suggested potential therapeutic or preventive strategies for managing HT, with vitamin D emerging as a candidate with immunomodulatory effects (4), and being proposed as a potential therapeutic option for HT (5).

Calcitriol, the active form of vitamin D, binds to vitamin D receptors (VDR) to regulate its interaction with the retinoic acid receptor (RXR) (6). RXR and VDR dimers are transported to the nucleus, where they bind to vitamin D-responsive elements (VDRE) and activate the transcription of target genes involved in various physiological processes (7). Vitamin D plays a major role in modulating immune and inflammatory responses by interacting with various immune cells (8). Macrophages are stimulated by calcitriol, which reduces the production of COX-2, and pro-inflammatory factors, while upregulating the production of anti-inflammatory interleukin (IL)-10 (9). Concomitantly, it has been suggested that reduced vitamin D is related to autoantibody production preceding autoimmune diseases due to its effects on B-cell differentiation, maturation, and proliferation (10). More direct evidence has been presented through in vitro tests using human B cells for the production of immunoglobulins against pyrogens (11). Vitamin D modulates CD4+ T lymphocyte activity by suppressing Th1 differentiation and promoting regulatory T cell development (12). It also influences dendritic cell-dependent T cell function and mitigates immune tolerance in thyroid tissues under autoimmune conditions (13).

Several studies have reported low levels of vitamin D and its converted forms (calcidiol and calcitriol) in patients with autoimmune diseases (10). Previous epidemiological studies have observed significant correlations among vitamin D deficiency, autoimmune thyroid disease, and elevated antithyroid antibody levels (13). The association between low vitamin D level and the occurrence of autoimmune diseases is well-established; however whether this deficiency is a cause or a consequence of the disease remains unclear (14, 15). Given the proven role of vitamin D in the regulation of immune responses under autoimmune conditions (8), researchers have investigated its direct effect on HT. A prospective randomized controlled clinical trial evaluating vitamin D supplementation in patients with newly diagnosed HT reported significantly attenuated thyroid marker of TPO-antibody (16). However, its direct effects on autoimmune thyroid diseases remain unclear.

To completely elucidate the clinically observed causal relationship between vitamin D deficiency and HT, practical laboratory investigations using patient samples are necessary. However, studies on biochemical changes at the cellular level in clinical thyroid samples from patients with HT are limited in number (17–19). No experimental study has demonstrated vitamin D-related mechanisms underlying the direct correlation between vitamin D or its signaling and HT pathology.

Transcriptomic profiling analysis, including RNA-seq and microarray analysis of the thyroid tissue, is a feasible method to address this problem (20). Using gene expression profiling data combined with bioinformatic tools and analyses, the overall pathophysiological changes in the HT cohort can be explored at the omics level (21). Gene set enrichment analysis (GSEA) can be performed to determine transcriptional activity in the pathway/biological process (BP) of interest in the analysis group (22). WikiPathways is an open-source pathway database that integrates pathway content from other primary pathway databases, including Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and Biopax, and provides broader options for analyzing vitamin D-related pathways (23).

We hypothesized that vitamin D deficiency and altered vitamin D signaling in thyroid tissue are associated with the development of HT. In this retrospective study, we investigated the potential relationship between vitamin D and HT by analyzing gene expression profiles obtained from thyroid tissue RNA-seq in Korean population. Using bioinformatic approaches, we mapped the gene expression profiles of patients with HT in various vitamin D-related wikipathways. Additionally, we validated the results using tissue histology and qPCR to confirm our hypothesis. This research will be useful for elucidating the dysregulation of the vitamin D signaling pathway in autoimmune thyroiditis and highlighting its potential as a therapeutic target. Our findings may also provide a molecular basis for considering vitamin D supplementation as a supportive strategy in HT management.

Several environmental and genomic factors reportedly play a role in the development of HT. In addition, a deep correlation between micro-nutritional factors, including iodine, selenium, and vitamins, and the occurrence of HT is recognized. Moreover, nutritional factors have regional characteristics based on food availability and surrounding environments (24). Vitamin D deficiency in the Korean population is a common problem, prevailing more frequently in women than in men, according to the Korea National Health and Nutrition Examination Survey conducted in 2008 (KNHANES) (25). This phenomenon may have a fundamental impact on autoimmunity-related conditions in the population. The HT cohort showed significantly lower serum vitamin D levels (especially 25(OH) vitamin D) ( Table 1 ).

Studies investigating the association between vitamin D levels and autoimmune thyroid diseases have been conducted. A previous study revealed an association between low vitamin D levels and HT in Korean population (26). This study classified patients with HT severity based on the hypothyroidism phenotype: euthyroid (ET), subclinical hypothyroidism (SCH), and overt hypothyroidism (OH). Comparing the vitamin D levels with the non-AITD group, the OH group showed significantly lower vitamin D levels (99.6 ± 53.7 vs 80.1 ± 47.7; p = 0.010). Conversely, a phenotype-centered case-control study showed a significantly higher prevalence of vitamin D insufficiency in patients with HT (27). A nationwide survey-based study elucidated the potential link between thyroid autoantibodies or thyroid dysfunction and insufficient vitamin D levels in women (28). A similar (reversal interaction) result between anti-TG levels and vitamin D was observed in our study in all participants (including all NT and HT groups in the statistical analysis). Although the negative correlation between serum vitamin D and anti-TG antibody levels was statistically significant, the very low R² (0.0248) indicates minimal explanatory power. Therefore, this finding should be interpreted with caution and considered hypothesis-generating rather than definitive.

The potential of vitamin D to treat autoimmune thyroiditis was evaluated by investigating the effects of vitamin D supplementation on thyroid antibody and hormone profiles, which resulted in a significant reduction in TG antibody levels (29). The therapeutic potential of vitamin D for the treatment of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, Crohn’s disease, and systemic lupus erythematosus, was evaluated by reviewing the results from clinical trials and only found inconsistent efficacy by studies (30). Strong evidence supporting the anti-inflammatory effects of vitamin D through the regulation of macrophage and T cell phenotypes may benefit HT patients by alleviating symptoms or, at the very least, preventing disease progression (30).

A meta-analysis of randomized controlled trials performed a combined evaluation of the efficacy of vitamin D treatment on thyroid function and autoimmune markers in patients with HT after a follow-up of three or six months (16). However, the meta-analysis found no significant association between serum vitamin D treatment and the levels of thyroid function and autoantibodies, except for a significant reduction in TPO-Ab levels (16). The inconsistent results of vitamin D supplementation studies highlight the need for a detailed consideration of factors, such as daily dosage, duration, and characteristics of the study cohort. Additionally, tissue-based laboratory analyses should be conducted to closely examine changes within the thyroid.

With the manifestation of the general clinical characteristics of HT by serum markers, activation of immunological responses and changes in thyroid function were significant in the HT group ( Figure 3 and Supplementary Table 3 ). While it was not on the top 12 list of BP terms, “response to vitamin D” was activated with most genes associated with the BP term significantly upregulated in HT ( Supplementary Table 3 ). This is the first report to demonstrate activation of the vitamin D signaling pathway in clinical tissue specimens of HT.

As a ligand-induced transcription factor complexed with RXR, VDR modulates the transcription of several genes (31). Dysfunction of the vitamin D receptor (VDR) has been proposed as a key factor in autoimmune diseases, as it degrades innate immune function (32). Previous reports on the correlation between VDR expression and vitamin D levels in pathological status have shown mixed and inconsistent results. An investigation of human VDR expression levels in ageing found no significant association between VDR expression and 25-hydroxyvitamin D (r = 0.002; p = 0.994) or 1,25-dihydroxyvitamin D (r = -0.108; p = 0.650) levels (33). In a study of patients with prostate cancer, significantly higher VDR expression levels were found in a group with a good prognosis, whereas no significant association was found between circulating vitamin D metabolite levels and VDR protein expression (34). A cohort study on sickle cell anemia reported elevated VDR expression in PBMCs of patients with vitamin D insufficiency (35). However, another study reported decreased VDR expression in T lymphocytes in the HT group, as demonstrated by qPCR analysis (36).

Our study revealed increased VDR expression and its related markers in the HT group. In addition, the expression of VDR-targeted genes showed mixed patterns due to the genomic action of vitamin D ( Figure 5A ). Consistently, reduced vitamin D levels and increased VDR expression were simultaneously observed in HT ( Supplementary Table 1 , Table 2 ), whereas the response to vitamin D was skewed toward activation ( Figures 4A, B ). Marshall’s model suggests that VDR dysfunction can explain the disrupted vitamin D analog levels (low 25-D (inactive form) and high 1,25-D (active form) serum levels) observed in a variety of autoimmune diseases (32). Several hypotheses could explain this paradoxical result including local activation of vitamin D by immune cells, ligand-independent VDR activation, and VDR gene polymorphisms that modulate its response to ligands.

In inflammatory diseases, the local levels of active vitamin D (1,25(OH)₂D) can be excessively elevated by activated macrophages and dendritic cells (37, 38), achieves local regulation of the vitamin D pathway at inflammatory sites by converting the inactivated vitamin D to active form (30). The pro-inflammatory status in our HT cohort was assumed to be due to the increased expression of transcription factors of inflammatory mediators, along with enhanced phospholipase (PLCs) and Protein Kinase C Alpha (PKAs) molecules ( Figure 5B ). In addition, histological examination revealed the diffuse boundary of the thyroid follicle structure in HT, which was oriented by an autoimmune response in the local tissue ( Figure 6A ). However, vitamin D can attenuate viral infection-induced inflammation and TLR3-mediated immune responses (39). Moreover, VDR on the macrophage membrane (VDRm) contributes to cell tolerance to LPS, suggesting the possibility of reducing inflammation through the suppression of innate immune responses.

VDR expression is regulated by LPS, which activates its sensing molecule, a member of the toll-like receptor (TLR) family (40). In monocytes, activated TLR and IFN-α induces autocrine and paracrine production of calcitriol by regulating CYP27B1 (41). In CD4+ T cells, calcitriol inhibits IFN-r production in Th1 cells and IL-4 production during Th17 cell differentiation and activation (8). Inhibition of vitaminD3 metabolism by blocking the CYP24A enzyme can enhance VDR signaling in cell level (42). Two genes participating in vitamin D activation and metabolism (CYP27B1 and CYP24A1) were slightly, however, not significantly, upregulated ( Figure 5D and Table 2 ). This may be due to the harvested tissue containing not only immune cells but also other types of cells from the thyroid tissue, making expression unclear.

The versatile biological activity of VDR-including genomic, non-genomic, ligand-dependent, and ligand-independent mechanisms- adds complexity to its interpretation (43). The ligand-independent function, similar to that of other nuclear hormone receptors, acts as a transcription regulator, with its specific roles varying across different cell types (44). The unliganded VDR exhibits non-classical actions by directly interacting with transcription factor or intracellular signaling molecules such as MAPK and PI3K which can have significant impact on innate and adaptive immunity (45, 46). Whether the increased vitamin D-related responses observed in the HT group are pathological characteristics or physiological adaptations to the activated immune response remains unclear.

Post-translational modifications (PTM) regulate VDR activity in diverse ways (47). Phosphorylation by PKC-β (Ser51) enhances transcriptional activation (48), while PKA-mediated phosphorylation (Ser182) reduces VDR–RXR interaction (49). In addition, ATM kinase and MAPK pathways (JNK1/p38) further modulate VDR, linking vitamin D signaling to immune regulation (50). These mechanisms highlight the importance of PTMs in shaping VDR function in autoimmune thyroid disease.

Another factor to consider in vitamin D signaling is VDR gene polymorphisms in trial subjects. Vitamin D receptor polymorphisms strongly affect vitamin D signaling (51) and are related to the susceptibility, severity, and development of several diseases (52). However, in some studies, the VDR polymorphism itself did not influence the expression level of VDR in patients with prostate cancer (34). Moreover, VDR polymorphisms were not involved in genetic susceptibility of the HT cohort, even though the prevalence of vitamin D deficiency was evident (70% vs. 18.2% for HT vs. control group; p < 0.0001) (53).

A limitation of this study is that we did not measure the active form of vitamin D (1,25(OH)₂D), which directly reflects local bioavailability and signaling. To fully understand the localized role of vitamin D in HT, the levels of its active form (1,25D) and the differences in the binding affinity of its ligand to VDR should be investigated in thyroid tissue and compared between NT and HT. In addition, further studies are needed to examine VDR polymorphisms to completely understand the etiology, susceptibility, and progression of the disease in relation to vitamin D signaling. Another limitation is that information on vitamin D supplementation was not available. Because supplementation could directly influence serum vitamin D levels, this represents a potential confounder in interpreting our findings (54). Future prospective studies should incorporate detailed records of supplement use to clarify its impact.

Our study included only female patients to reduce variability related to sex hormones and immune responses. However, this design limits the generalizability of our findings to male patients, in whom vitamin D metabolism and immune regulation may differ. Future studies including both sexes are warranted to clarify potential sex-specific differences in the relationship between vitamin D signaling and autoimmune thyroiditis. In addition, because both HT and NT tissues were obtained from patients undergoing surgery for thyroid neoplasms, tumor-related microenvironmental influences on vitamin D signaling could not be completely ruled out. This potential confounder should be considered when interpreting the results. The small RNA-seq sample size limits the statistical power and generalizability, so these results should be considered exploratory and hypothesis-generating pending validation in larger cohorts.

Our study presents the first RNA-seq dataset obtained from a Korean HT cohort, and elucidates the dysregulation of vitamin D and its signaling in HT. Through integrated research, we found that the HT population exhibits upregulated expression of VDR and other genes associated with vitamin D-related pathways in the thyroid tissue, despite low vitamin D levels in serum. However, the present study design does not allow for a conclusion as to whether vitamin D deficiency contributes to, or results from, the development of HT. Further studies are warranted to clarify the causal relationship between vitamin D deficiency and the development of HT, and should incorporate functional approaches such as measurement of local 1,25(OH)₂D levels in thyroid tissue, assessment of VDR binding and transcriptional activity, and analysis of VDR gene polymorphisms.