HT, also known as chronic lymphocytic thyroiditis, is the most common autoimmune thyroid disease, accounting for approximately 22.5% of thyroid diseases (72). It is characterized by infiltration of thyroid-specific T lymphocytes and other immune cells, goiter enlargement and fibrosis, destruction of thyroid cells, and eventually hypothyroidism (73). A few HT patients have no goiter, and approximately 50% of patients have clinical hypothyroidism. The characteristic autoantibodies of HT mainly include TPOAb and TGAb (74), which have the function of fixing complement, causing cytotoxicity and damaging thyrocytes. In addition, HT patients may also have thyroid stimulating blocking antibodies (TSBAbs) (74), which can promote thyroid atrophy and hypofunction. In HT patients, Th1 cells are the dominant infiltrating lymphocytes, which release cytokines (IFN-γ, IL-2, and TNF-α) under the stimulation of thyroid autoantigen, and the latter stimulates the expression of Fas on the surface of the thyroid cells, thereby accelerating the apoptosis of the thyroid cells (75). Exosomes from human B cell-derived lymphoblastoid cell lines highly express MHC II and FasL, which can induce the apoptosis of CD4⁺ T cells through the interaction of FasL and Fas (40, 76, 77). Therefore, exosomes could be associated with cell apoptosis and participate in several pathological processes related to HT.

Exosomal surface protein markers such as HSP60 and HSP70 can mediate immunomodulatory effects and immune responses (78, 79). HSP60 can bind a variety of receptors present on the surface of immune cells, such as TLRs, CD40, and CD9, leading to antigen cross-presentation, T cell cross-priming, and immune response (78, 80). Cui et al. (81) detected serum exosomes of 40 HT patients and 40 healthy controls, and concluded that exosomes from HT patients (HT-Exos) highly expressed the inflammatory factor HSP60, which was positively correlated with serum TPOAb and TgAb. After coculture with healthy human DCs, HT-Exos derived HSP60 was spatially closely arranged and more fully bound to TLR2 on DCs compared with intracellular HSP60. Similarly, another study found that the level of HSP60 in thyroid follicular cell-derived exosomes stimulated by IFN-γ was significantly higher than that in normal thyrocyte-derived exosomes. Interestingly, the structure of HSP60 is highly homologous with the structures of TPO and TG, and mediates the disruption of thyrocytes through antigen-antibody reactions in HT (82). Therefore, there is a hypothesis that exosomal HSP60 may serve as an antigen to mediate immunological functions in HT pathogenesis. However, further studies are required to identify the mechanisms of exosomal HSP60-mediated regulation of HT pathogenesis.

Current studies have demonstrated that Th1/Treg, Th2/Treg and Th17/Treg cells in HT patients are out of balance at the transcription factor level, manifesting as the upregulation of Th1, Th2 and Th17 cells, and the downregulation of Treg cells (83–85). In this regard, exosomes from HT patients cocultured with peripheral blood mononuclear cells (PBMCs) of healthy humans increased the percentage of CD4⁺ IFN-γ⁺ Th1 cells and CD4⁺IL-17A⁺ Th17A cells in PBMCs, and decreased the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in PBMCs (81). Furthermore, this study also found that exosomes from HT patients cocultured with DCs bind to TLR2/3 of DCs, and activate DCs through the TLR2/myeloid differentiation factor 88 (MyD88)/NF-κB and TLR3/TRIF/NF-κB pathways, thus leading to an imbalance in the differentiation of CD4⁺ T lymphocytes. In conclusion, these findings support a possible role for exosomes in HT pathogenesis: the levels and composition of circulating exosomes in HT patients could induce T cell imbalance.

As the most powerful professional APCs and CD4⁺ T lymphocytes, DCs are the main cells involved in the inflammatory response, and they participate in the pathogenesis of HT (86). Research has revealed that APC-derived exosomes play a role in antigen presentation and T lymphocyte activation. For example, mature DC-derived exosomes have been shown to induce the proliferation of CD4⁺ T cells in an antigen-specific manner (87), but the role of exosomes derived from nonprofessional APCs (such as thyroid follicular cells) in these processes is rarely described. Another study by Cui et al. (88) found that thyroid follicular cell-derived exosomes stimulated by IFN-γ (IFN-γ-Exos) cocultured with DCs increased the expression of costimulators CD40 and CD80 and the mature marker CD83 on DCs, and increased the gene expression levels of inflammatory cytokines such as IL-6 and TNF. Furthermore, after culturing DCs, IFN-γ-Exos cocultured with CD4⁺ T lymphocytes from healthy humans significantly increased the mRNA expression levels of IFN- γ, IL-17A, IL-22, IL-4, IL-10 and TGF-β1 in CD4⁺ T cells. All of the evidence suggests that exosomes could activate DCs to further cause an imbalance in cytokine expression and the secretion of CD4⁺ T cells, which may lead to the formation of thyroid tissue and a systemic inflammatory environment. These inflammatory microenvironments can further promote the release of “abnormal exosomes” from thyrocytes in vivo, forming a positive feedback loop involved in HT pathogenesis. Additionally, another study observed that the exosome inhibitor GW4869 blocked the activation of DCs by thyroid follicular cells after inhibiting IFN-γ-Exos, suggesting that exosomes play a key role in antigen presentation and the inflammatory response. GW4869 is a neutral sphingomyelinase inhibitor with cell permeability and selectivity, and it is basically recognized as an exosome inhibitor. It can inhibit the secretion of exosomes in vivo or in vitro, which has been reported in tumors, inflammation and infection (89–92). These studies indicate that intervention in exosome secretion may have a therapeutic effect on inflammation and autoimmune diseases. Together, these results suggest a potential role of thyroid follicular cell-derived exosomes in regulating T cell differentiation imbalance in the occurrence and development of HT, but the specific mechanism remains to be further clarified.

GD, namely, diffuse toxic goiter, is an organ-specific autoimmune disease with abnormal thyroid hormone synthesis, accounting for more than 85% of patients with hyperthyroidism, with a high incidence in women aged 40-60 years old (93). GD mainly manifests as thyrotoxicosis, diffuse goiters and Graves’ ophthalmopathy (GO) (94). Abnormal infiltration of T lymphocytes and production of the autoantibody TRAb are the pathophysiological basis of GD (95). The imbalance of Th1/Th2 cells has become a research focus of GD in recent years. It is generally believed that the inflammatory response mediated by Th1 cells is dominant in the active phase of GD, while Th2 cells mainly play a role in the inactive phase of GD (96). In GD, the recruitment of Th1 lymphocytes leads to increased production of IFN-γ and TNF-α, which stimulates thyroid cells to secrete Th1 chemokines, promoting the autoimmune process. A study (97) investigated the role of IL-21 in the regulation of Th17/Treg cells in 28 newly diagnosed GD patients, 27 remission GD (EGD) patients and 24 healthy subjects and found that IL-21 could stimulate the differentiation of CD4⁺ T cells into Th17 cells, reduce the differentiation of Treg cells, and contribute to the activation of the downstream immune response and the pathogenesis of GD.

A number of studies have implicated that IGF-1R and TSHR (thyrotropin receptor) are involved in GD pathogenesis and have a direct interaction in thyroid related eye diseases. Reports from Huang et al. (98) demonstrated that IGF-1R, TPO, TSHR and HSP60 existed in serum exosomes from GD patients and healthy controls. In particular, the levels of IGF-1R and TPO in the serum exosomes of GD patients and active GO patients without hormone shock therapy were significantly higher than those in healthy controls. Further correlation analysis showed that IGF-1R was significantly positively correlated with TRAb levels, suggesting that exosomes may present antigens and carry autoantigens (IGF-1R) in GD pathogenesis. However, further study is required to identify why circulating exosomes are specifically recognized as autoantigens in GD.

Additionally, Rossi and his colleagues observed that the formation and transfer of exosomes from thyroid follicular cells induced by dichlorodiphenyltrichloroethane (DDT) contained thyrotropin-releasing hormone, which could directly stimulate TSHR to binding to autoantibodies, leading to GD development (99). Interestingly, Edo et al. (100) provided evidence that TSHR is detected in exosomes secreted by normal thyroid follicular cell lines and cancerous thyroid cell lines. To examine the function of TSHR exosomes in GD, HEK (human embryonic kidney)/TSHR cells were generated as a model for thyroid follicular epithelial cells in GD where TSHR is upregulated. This study demonstrated that TSHR exosomes isolated from HEK/TSHR cells may exert a decoy effect by binding to and sequestering autoantibodies, thereby ameliorating autoantibody-mediated activation of thyroid function in a GD model. These results seem to contradict each other; therefore, it is necessary to further study the function of TSHR in exosomes in GD.

Circulating exosomes from GD patients were observed to be immunologically active and were capable of inducing the production of inflammatory cytokines from healthy PBMCs. Hiratsuka et al. (101) isolated serum exosomes from patients with refractory GD, and these exosomes stimulated the mRNA expression of inflammatory cytokines such as IL-1, IL-6 and TNF-α in PBMCs, thereby activating the immune response over that found in GD patients in remission or healthy controls. Similarly, Huang et al. (98) uncovered that coculture of serum exosomes from GD patients and healthy human PBMCs promoted the increased expression of CD11c⁺TLR2⁺ DCs and CD11c⁺TLR3⁺ DCs in PBMCs as well as an increase of IL-6 and IL-1β in the supernatant. These results suggest that exosomes from GD patients are able to promote the inflammatory response, favoring the pathogenesis and development of GD. To further investigate the specific mechanism of exosomes in promoting the inflammatory response, Cui et al. (102)recently analyzed serum exosomes from 26 healthy controls (HC-Exos), 26 GD patients (GD-Exos) and 7 Graves ophthalmopathy patients (GO-Exos). In this study, after coculture of healthy human PBMCs with GD-Exos or HC-Exos for 24 hours, GD-Exos could bind to TLR2/3 and increase the percentage of CD11c⁺TLR2⁺ DCs and CD11c⁺TLR3⁺ DCs in PBMCs; therefore, GD-Exos may mediate their role by acting on TLRs. Furthermore, GD-Exos also significantly increased the protein expression of MyD88, TRIF and p-P65 in PBMCs, as well as the levels of IL-6 and IL-1β in the medium. It is therefore possible that GD-Exos might participate in GD pathogenesis by binding to TLR2/3 to activate the NF-κB signaling pathway and induce an inflammatory response. While the mechanisms have not been fully revealed, these observations provide clues that exosomes can promote the inflammatory response in GD.

In addition to synergies between immune factors and environmental stimulation, epigenetics also plays an important role in the pathogenesis of GD, including DNA demethylation, histone modification and noncoding RNAs(ncRNAs) interference (103, 104). Intriguingly, the role of noncoding RNAs (including miRNAs, long noncoding RNAs, and circRNAs) in the etiopathogenesis of GD is increasingly attracting attention (105, 106). It has been revealed that nucleic acids contained within exosomes retain their function after exosomal vesicles direct transfer their contents into a recipient cell (107). Research has discovered differences in exosomal noncoding RNAs between healthy populations and GD patients at different stages. Hiratsuka et al. (101) indicated that plasma exosomes from GD patients carry upregulated or downregulated miRNA levels and are associated with disease progression. In this study, compared with intractable GD patients, the levels of miR-23b-5p and miR-92a-3p in serum exosomes of GD patients in remission were significantly increased, while the levels of let-7g-3p and miR-339-5p were significantly decreased. However, the molecular mechanisms of these miRNAs related to GD have not yet been fully elucidated. Other studies have discovered that miRNAs in serum exosomes of intractable GD patients can stimulate mRNA expression of IL-1β and TNF-α, which may be closely related to the pathogenesis and disease progression of GD. Additionally, Sun et al. (108) showed that plasma exosome circRNAs from GD patients were more than twice as upregulated or downregulated compared with healthy controls, with 15 circRNAs significantly differentially expressed, including the upregulation of 6 circRNAs and the downregulation of 9 circRNAs. This study further verified the significantly increased expression level of has-circRNA-000102 in the plasma exosomes of GD patients and revealed the potential pathways related to GD pathogenesis, namely, herpes simplex virus infection, influenza A signaling and IFN-β signaling. These observations imply the potential roles of exosome-derived noncoding RNAs as significant regulators and biomarkers for diagnosing GD. However, more studies are required to investigate exosomal RNAs and their significance in the pathophysiology and diagnosis of GD.

Thyroid ophthalmopathy (TED) is the most common extrathyroid manifestation of AITD. It includes GO, which in severe cases can result in visual field loss due to corneal rupture or compression optic neuropathy (109). Han et al. (110) isolated tear exosomes from GD patients with TED, and found that the tear exosomes of TED patients were 2.3 times higher than those of healthy controls, and highly expressed vitamin D binding protein (VDBP), C-reactive protein (CRP), chitinase 3-like 1 (CH3L1), matrix metalloproteinase-9 (MMP-9) and vascular adhesion molecule-1 (VCAM-1).

Matrix metalloproteinases play a key role in tissue remodeling in the process of fibrosis and inflammation. They are expressed at very low levels in normal tissues, but can be increased by the influence of inflammatory cytokines, intercellular interactions, hormones, and growth factors (111). Tear derived exosomes can trigger orbital fibroblasts to release the inflammatory cytokines, IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP1) in vitro. These findings suggest that an increased abundance of specific proteins in exosomes can also activate inflammatory responses through orbital fibroblasts (the target cells in TED). In general, increased levels of MMP-9 in tear exosomes could promote orbital tissue remodeling and fibrosis, which contributes to the occurrence and development of TED.