Abstract
Experimental models of hyperthyroid Graves' disease (GD) and Graves' orbitopathy (GO) are efficiently developed by genetic immunisation by electroporation with human thyrotropin hormone receptor (hTSHR) A-subunit plasmid in female BALB/c (H-2d) mice. We investigated susceptibility in C57BL/6 J (H-2b) animals to allow studies on disease mechanisms in transgenic and immune response gene knock-out mice. Higher numbers of female C57BL/6 J were positive for pathogenic thyroid stimulating antibodies, but induced hyperthyroidism remained at a low frequency compared to BALB/c animals. Assessment of hTSHR specific T cells showed reduced proliferation in C57BL/6 J animals accompanied with anti-inflammatory IL-10, with less pro-inflammatory IFN-γ compared to BALB/c. Whilst the orbital tissue from immune BALB/c mice showed inflammation and adipogenesis, in contrast C57BL/6 J animals showed normal pathology. We characterised the gut microbiota using 16 S ribosomal RNA gene sequencing to explore its possible pathogenic role in the model. Despite being housed under identical conditions, we observed significantly different organisation of the microbiota (beta-diversity) in the two strains. Taxonomic differences were also noted, with C57BL/6 J showing an enrichment of Operational Taxonomic Units (OTUs) belonging to the Paludibacter and Allobaculum, followed by Limibacter, Anaerophaga and Ureaplasma genera. A higher number of genera significantly correlating with clinical features was observed in C57BL/6 J compared to BALB/c; for example, Limibacter OTUs correlated negatively with thyroid-stimulating antibodies in C57BL/6 J mice. Thus, our data suggest gut microbiota may play a pivotal immunomodulatory role that differentiates the thyroid function and orbital pathology outcome in these two inbred strains undergoing experimental GO.
Highlights
Variation in induced models of autoimmunity has been attributed to the housing 3 environment and its effect on the gut microbiota
We found an enrichment of Operational Taxonomic Units (OTUs) of Acetitomaculum genus in the βgal group compared to both TSHR-immunized mice (TSHR) (P=0.004) and the untreated group (P=0.003); an enrichment of Lactobacillus OTUs in the TSHR compared to the untreated group (P=0.018) and a reduction of Bacteroides counts in TSHR when compared to the βgal group (P=0.047)
Correlating the gut microbiota composition with clinical features and differences in Graves’ orbitopathy (GO) development We investigated possible correlations between disease features, such as anti-TSHR antibodies, thyroxine levels, orbital adipogenesis and muscular atrophy, and the gut microbiota composition to determine whether it contributes to the heterogeneity of induced responses, summarized in Additional file 1: Table S1
Summary
Variation in induced models of autoimmunity has been attributed to the housing 3 environment and its effect on the gut microbiota. The intestinal monocolonization of germ free mice (sterile) with segmented filamentous bacteria (SFB) restored the disease phenotype, along with an increased number of Th17 cells in the CNS, suggesting a direct interplay of the gut microbiota and the immune response in EAE development [20]. Based on these observations, we hypothesized that the gut microbiota itself might play a major role in the establishment, and in the reproducibility of the GO animal model described above. We characterized the gut microbiota between different immunizations (TSHR and βgal) and compared them with untreated mice, to determine whether the gut microbiota can influence the outcome and correlate with disease features
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