Comparative assessment of female mouse model of Graves' orbitopathy under different environments, accompanied by pro-inflammatory cytokine and T cell responses to thyrotropin hormone receptor antigen

We recently described a preclinical model of Graves' orbitopathy (GO), induced by genetic immunization of eukaryotic expression plasmid encoding human TSH-receptor (hTSHR) A-subunit by muscle electroporation in female BALB/c mice. The onset of orbital pathology is characterized by muscle inflammation, adipogenesis and fibrosis. Animal models of autoimmunity are influenced by their environmental exposures. This follow-up study was undertaken to investigate the development of experimental GO in two different locations, run in parallel under comparable housing conditions. Functional antibodies to TSHR were induced in TSHR A-subunit plasmid immunized animals, and antibodies to IGF-1 receptor α subunit were also present, while control animals were negative in both locations. Splenic T cells from TSHR A-subunit primed animals undergoing GO in both locations showed proliferative responses to purified TSHR antigen and secreted IFN-γ, IL-10, IL-6 and TNF-α cytokines. Histopathological evaluation showed orbital tissue damage in mice undergoing GO, manifest by adipogenesis, fibrosis and muscle damage with classic signs of myopathy. Although no inflammatory infiltrate was observed in orbital tissue in either location, the appearances were consistent with a 'hit and run' immune-mediated inflammatory event. A statistically significant increase of cumulative incidence of orbital pathology when compared to control animals was shown for both locations, confirming onset of orbital dysimmune myopathy. Our findings confirm expansion of the model in different environments, accompanied with increased prevalence of T cell derived pro-inflammatory cytokines, with relevance for pathogenesis. Wider availability of the model makes it suitable for mechanistic studies into pathogenesis and undertaking of novel therapeutic approaches

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Additional file 6: of Gut microbiota in experimental murine model of Graves’ orbitopathy established in different environments may modulate clinical presentation of disease

Figure S1. NMDS plot based on the weighted Unifrac distances of Center2 immune and control mice including TSHR-immunized mice from Center 1. TSHR-immunized mice from Center 1 were more similar to TSHR-immunized mice from Center 2 (P = 0.2) than to the βgal (P = 0.024) than the untreated (P = 0.04). (PDF 28 kb

Additional file 2: of Gut microbiota in experimental murine model of Graves’ orbitopathy established in different environments may modulate clinical presentation of disease

Supplementary methods. (DOCX 121 kb

Additional file 1: of Gut microbiota in experimental murine model of Graves’ orbitopathy established in different environments may modulate clinical presentation of disease

Figure S1. Schematic representation of the GO immunization protocol and sample collection. Table S1. Summary of disease characteristics induced in mice in Center 1 and Center 2 using TSHR expression plasmid illustrating the heterogeneity of response. Table S2. Quarterly Health Screen Reports on viral, bacterial, mycoplasma and parasite screen in both centers. Table S3. Composition of the commercial chows provided ad libitum in Center 1 and Center 2. (DOCX 106 kb

Additional file 3: of Gut microbiota in experimental murine model of Graves’ orbitopathy established in different environments may modulate clinical presentation of disease

Table S1. Differential abundant taxonomic analysis between TSHR (n = 10), βgal (n = 8), and untreated (n = 6), within Center 2. Welch’s T-test with 95% confidence interval using STAMP. Mean relative frequency, rel. freq. Standard deviation, std. dev. Table S2. Comparison of intestinal scraped samples from different immunization within Center 2 from the traditional microbiological culture. Data were Box-Cox transformed. (XLSX 43 kb