Graves’ disease is a common autoimmune thyroid disorder, but the role of infection-related immune responses in its development remains unclear. This study is important because it examines whether antibody responses to common infectious agents are likely to contribute to Graves' disease, or whether Graves’ disease itself may influence these immune responses. By analyzing large genetic datasets in both directions, the study provides evidence that antibody responses to infections may not directly cause Graves’ disease. Instead, genetic susceptibility to Graves’ disease appears to be associated with altered antibody responses to cytomegalovirus, Helicobacter pylori, and polyomaviruses. These findings add new evidence to the literature by shifting attention from infection as a simple trigger toward Graves' disease as a condition that may reshape immune responses to infection. This may help guide future studies on immune monitoring and infection-related care in Graves’ disease.

Graves’ disease (GD) is a common organ-specific autoimmune disorder, a subtype of autoimmune thyroid disease (AITDs), characterized by hyperthyroidism and most commonly seen in women aged 30–60 years [1]. The pathophysiology involves multiple immunoregulatory factors, including immune responses mediated by thyroid autoantibodies. Patients with GD have abnormal stimulatory autoantibody against the thyroid-stimulating hormone receptor (TSHR). These autoantibodies bind to TSHR to activate the adenylate cyclase signaling system, leading to diffuse goiter, hyperthyroidism, and infiltrative ophthalmopathy. The disease reflects a complex interaction between genetic susceptibility, environmental factors, and immune responses, particularly TRAb [2–4]. In recent years, the incidence of this disease has been rising, and the average age of patients is decreasing [5]. The primary treatment modalities include anti-thyroid drugs (ATD), radioactive iodine (RAI), and thyroidectomy. However, these approaches tend to have low remission rates, high recurrence rates, and numerous complications [6, 7].

Antibody-mediated immune response is a key mechanism to defend against infection by pathogenic microorganisms. Many infectious agents may play a role in the onset and progression of noncommunicable diseases (NCDs), especially various autoimmune disorders [8–10]. The onset of AITD may be triggered by infectious agents [11]. Patients with GD might exhibit different response patterns to certain infections due to the persistent high activity of their immune systems. For instance, studies have shown that some patients with GD have positive serum HCV tests [12]. Previous observational and mechanistic studies have suggested that infectious agents may contribute to the development of AITD, although the causal direction and the underlying immune pathways remain uncertain [13].

Mendelian randomization (MR) is a statistical method that uses genetic variants as a tool to assess the causal relationship between risk factors and disease [14]. To uncover the potential causal relationship between the immune response mediated by anti-infective agent antibodies and GD, we conducted a study using two-way Mendelian randomization. Using gene variants as instrumental variable, this method can effectively avoid the confounding factors and reverse causation problems that exist in the traditional observational studies and, thus providing more reliable causal inference. MR avoids the limitations of randomized controlled trials such as ethical limitations, high cost and long duration, and gradually becomes a popular analysis method.

Through this study, we aim to enhance our understanding of the role of antibody-mediated anti-infective immune response in the pathogenesis of GD and explore their potential applications in clinical interventions and treatments for GD.

In the forward MR analysis, none of the infection-related antibody-response phenotypes was associated with GD risk after Bonferroni or FDR correction, although three Epstein-Barr virus antibody phenotypes showed nominal inverse associations. In the reverse MR analysis, genetic liability to GD was associated with selected antibody-response phenotypes. Among the five prioritized reverse associations, the association with anti-polyomavirus 2 IgG seropositivity remained significant after FDR correction, whereas the other four associations were suggestive. These findings should therefore be interpreted at the level of genetically predicted antibody-response traits rather than as evidence of an established immunological mechanism.

Current studies have shown that the development of autoimmune diseases may be related to infection [20–22]. Infection with specific pathogens can trigger or worsen autoimmune responses, especially when combined with genetic predisposition and environmental factors. In some prospective cohort studies and observational studies, immune response to Epstein-Barr virus (EBV) is closely associated with susceptibility to SLE [23], as well as with the pathogenesis and poor control of rheumatoid arthritis (RA) [24]. Elevated levels of EBV EBNA-1 antibodies were also observed in the sera of patients with polymyositis (PM), multiple sclerosis (MS) [25], and Hashimoto’s thyroiditis [26]. HLA class I and immune signaling proteins, such as STAT1 and PKR(which are involved in antiviral responses), have been found to be upregulated in thyroid tissues of GD patients, suggesting that viral infection may initiate or exacerbate the autoimmune process [27]. These studies provide biological context for the relationship between infection and autoimmunity. However, they address a different question from the present MR analysis, which evaluates whether genetically predicted lifelong differences in antibody-response phenotypes are associated with GD risk and whether genetic liability to GD is associated with these phenotypes.

Cytomegalovirus (CMV) is a common DNA virus that lurks in human tissue after infection and can reactivate in cases of immune deficiency, causing herpes [28]. CMV pp52 antibody levels are often used to assess the presence of active CMV infection in the body. Studies have found higher levels of pp52 protein IgG and IgA antibodies against CMV in patients with systemic lupus Erythematosus (SLE) [29]. However, CMV pp52 IgG levels are significantly reduced in patients with multiple sclerosis [30]. Consistent with this observation, our reverse MR analysis showed that genetic liability to GD was nominally associated with lower CMV pp52 antibody levels. These findings suggest that autoimmune disease susceptibility may be associated with alterations in CMV-related humoral immune responses and provide a basis for further investigation. Protein pp150 is related to the maturation and release of CMV and has strong immunogenicity [31]. A study on human cytomegalovirus (hCMV) has shown that anti-PP150 antibodies associated with multiple autoimmune diseases can recognize the viral pp150 protein and the human CIP2A protein, resulting in CD56 (bright) NK cell death [32]. The decrease in the number and function of NK cells is a common pathogenesis of many autoimmune diseases. Some studies have shown that active CMV infection in the synovial fluid of RA patients may exacerbate the inflammatory process. The presence of pp150 also suggests a potential reactivation of CMV in these patients, which could be linked to the pathological processes of rheumatoid arthritis (RA) [31].

Helicobacter pylori (H. pylori) is a bacterium associated with gastritis, stomach ulcers, and certain types of gastric cancer [33]. In addition, its infection has been linked to various extra-gastric diseases [34], especially the development of AITD, including Hashimoto’s thyroiditis (HT) and GD [21, 35–37]. Studies have shown that antibodies against H. pylori can cross-react with thyroid tissue [38]. In one study, researchers used a stool antigen test to analyze Helicobacter pylori infection, confirming a significant correlation between this bacterium and patients with GD. In GD patients, the detection rate of Cag-A positive H. pylori strain was significantly higher [37]. This may be related to antibodies produced during Helicobacter pylori infection, and antibodies against the Cag-A protein can cross-react with thyroid antigen, potentially triggering the development of GD [21]. Urease is a specific enzyme of this bacterium that helps neutralize stomach acid and is closely related to the bacterium’s stress response [39]. There is evidence of common epitopes between thyroid follicular epithelial and gastric parietal cells [40]. Some studies have suggested that proteins from H. pylori, including urease, may share epitopes with thyroid follicular cells, potentially leading to molecular mimicry and triggering an autoimmune response [41]. In the present reverse MR analysis, genetic liability to GD was nominally associated with higher H. pylori UreA antibody levels. This finding suggests a potential association between GD susceptibility and the H. pylori-related humoral immune response. However, molecular mimicry and antibody cross-reactivity remain possible explanations derived from previous studies and require functional validation in the context of GD.

Polyomavirus (PyVs) is a small DNA virus that causes tumor diseases in humans and other animals [42]. Merkel-cell polyomavirus is known to cause invasive skin cancer of neuroendocrine origin, with a higher risk in immunosuppressed individuals [43]. SV40(Simian virus 40), belonging to the polyomavirus 50 genus, was found in thyroid tissue samples from 20% of GD patients. Although this ratio is low, it suggests that SV40 may be associated with the development of autoimmune thyroid diseases [44]. In our reverse MR analysis, the association between genetic liability to GD and anti-polyomavirus 2 IgG seropositivity remained significant after FDR correction, although it did not reach the Bonferroni-corrected threshold. A nominal association was also observed for anti-Merkel cell polyomavirus IgG seropositivity. These findings support further investigation of the relationship between GD susceptibility and polyomavirus-related antibody responses, while the involvement of viral persistence, reactivation, or thyroid-local infection remains to be established.

Of course, this study has some limitations, First, although the pooled GWAS data of European ancestry were used to reduce ethnic bias, the generalizability of the results across different populations remains uncertain. Further GWAS studies with larger samples are needed to update the results. Second, Only studies of the risk between GD data and antibody-mediated immune responses in the Finnish database have been conducted, and the risk between other GD data and antibody-mediated immune responses in the database has not been explored. Finally, the antibody phenotypes analyzed in this study are proxies for humoral immune responses and may not fully reflect acute pathogen exposure, the timing or reactivation of infection, cellular immunity, or local immune responses within thyroid tissue. Because MR estimates the effects of genetically predicted lifelong differences in these traits, complementary longitudinal and mechanistic studies are needed to further clarify the relationship between infection and GD.

In summary, our bidirectional MR analysis suggest that antibody-mediated immune responses to infections may not directly cause GD. Instead, the development of GD pathology may be an indirect consequence of these immune responses, or possibly affected by other unknown factors. A deeper understanding of their complex interactions lays the groundwork for further research and potential therapeutic strategies.