A Promising Mouse Model of Graves' Orbitopathy Induced by Adenovirus Expressing Thyrotropin Receptor A Subunit

Abstract

Background: Graves' orbitopathy (GO) is the most common and serious manifestation of Graves' disease (GD). It is characterized by orbital inflammation and tissue remodeling. Although several GO models have been reported, most lack a full assessment or mechanistic evaluation. Here, we established a promising mouse model mimicking many aspects of human GO with a frequency of 70% and characterized the key role of T cells in the progression of GO. Methods: An adenovirus expressing the human thyrotropin (TSH) receptor A subunit (Ad-TSHRA) was injected in the muscles of female BALB/C mice nine times to induce GO. At predetermined time points, histological examinations of retrobulbar tissues and thyroid glands were performed to dynamically monitor changes; serum autoantibodies and total thyroxine levels were examined to evaluate thyroid function. Flow cytometry of CD4+ T cell subgroups and RNA sequencing (RNA-Seq) of splenocytes were also performed to explore the underlying mechanism. Results: After nine injections, 7 of 10 mice challenged with Ad-TSHRA developed the orbital changes associated with GO. Seven mice manifested retrobulbar fibrosis, and four mice showed adipogenesis. Exophthalmia, conjunctival redness, and orbital lymphocyte infiltration were also observed in a subset of mice. The orbitopathy was first detected after seven injections and followed the hyperplastic change observed in thyroids after four injections. Flow cytometry revealed increased proportions of Th1 cells and decreased proportions of Th2 cells and regulatory T (Treg) cells in the splenocytes of GO mice. This change in CD4+ T cell subgroups was confirmed by orbital immunohistochemical staining. Genes involved in T cell receptor signaling, proliferation, adhesion, inflammation, and cytotoxicity were upregulated in GO mice according to the RNA-Seq; a trend of upregulation of these GO-specific genes was observed in mice with hyperthyroidism without orbitopathy after four injections. Conclusions: A GO mouse model was successfully established by administering nine injections of Ad-TSHRA. The model was achieved with a frequency of 70% and revealed the importance of T cell immunity. A potential time window from Graves' hyperthyroidism to GO was presented for the first time. Therefore, this model could be used to study the pathogenesis and novel treatments for GO.

Mechanisms That Underly T Cell Immunity in Graves’ Orbitopathy

“…Evidence from animal models is indicating that IL-17A and IFN-γ double-producing Th17 cells are pathogenic drivers of various human autoimmune diseases such as multiple sclerosis, diabetes type 1, uveitis, dry eye, rheumatoid arthritis, and inflammatory bowel disease ( 100 , 114 ). Unfortunately, no convincing evidence of detectable Th17 cells has been observed in current GO murine models ( 36 , 53 ), which makes it difficult to prove our hypothesis. The distinctive genetic backgrounds of BALB/c and C57BL/6J mice may partially be responsible for their susceptibility to GD and GO as well as the different T cell responses under autoimmune disease conditions ( 116 ).…”

“…In hTSHR-A subunit plasmid-primed GO BALB/c mice, splenic T cells secreted IFN-γ ( 53 ). An increased frequency of CD4 + IFN-γ-producing T cells derived from splenocytes has also been observed in hTSHR-A subunit-expressing adenovirus-immunized GO BALB/c mice ( 36 ). In an SKG murine model of GO, expression of Ifng , Tnfa , and Il2 was augmented in periorbital tissues and their protein levels were elevated in sera compared with wild type mice ( 48 ).…”

“…Intriguingly, increased CD4 + T cell subsets were reported in periorbital fat of SKG mice after intraperitoneal administration of zymosan A compared with wild type mice ( 48 ). A recent study used an adenovirus that expressed the hTSHR-A subunit to induce GO in BALB/c mice and also observed CD4 + T cell infiltration in periorbital fat tissues ( 36 ). Collectively, these data shed light on the presence and type of T cells in GO, which suggest a complex inflammatory microenvironment in the orbit.…”

“…Studies in GO murine models have not been able to duplicate Th2-dominated immune responses. A decreased frequency of CD4 + IL-4-producing splenic T cells has been observed in hTSHR-A subunit-expressing adenovirus-immunized GO BALB/c mice ( 36 ). However, compared with wild type mice, expression of Il4 , Il5 , and Il13 was increased in periorbital tissues of GO SKG mice ( 48 ).…”

Insight Into Mouse Models of Hyperthyroidism

“…However, the conclusion was based on an analysis of the short-term adenovirus induction protocols. Studies have shown that prolonging the induction time of adenovirus expressing the TSHR (ad-TSHR) A subunit can increase the percentage of successful induction of GO (37). Zhang et al injected an adenovirus expressing the human TSHR A subunit into the muscle of female BALB/c mice 9 times and successfully induced GO model after long-term ad-TSHR A subunit immunization (37).…”