Gut microbiota in experimental murine model of Graves' orbitopathy established in different environments may modulate clinical presentation of disease

Abstract

Background: Variation in induced models of autoimmunity has been attributed to the housing environment and its effect on the gut microbiota. In Graves' disease (GD), autoantibodies to the thyrotropin receptor (TSHR) cause autoimmune hyperthyroidism. Many GD patients develop Graves' orbitopathy or ophthalmopathy (GO) characterized by orbital tissue remodeling including adipogenesis. Murine models of GD/GO would help delineate pathogenetic mechanisms, and although several have been reported, most lack reproducibility. A model comprising immunization of female BALBc mice with a TSHR expression plasmid using in vivo electroporation was reproduced in two independent laboratories. Similar orbital disease was induced in both centers, but differences were apparent (e.g., hyperthyroidism in Center 1 but not Center 2). We hypothesized a role for the gut microbiota influencing the outcome and reproducibility of induced GO.

Results: We combined metataxonomics (16S rRNA gene sequencing) and traditional microbial culture of the intestinal contents from the GO murine model, to analyze the gut microbiota in the two centers. We observed significant differences in alpha and beta diversity and in the taxonomic profiles, e.g., operational taxonomic units (OTUs) from the genus Lactobacillus were more abundant in Center 2, and Bacteroides and Bifidobacterium counts were more abundant in Center 1 where we also observed a negative correlation between the OTUs of the genus Intestinimonas and TSHR autoantibodies. Traditional microbiology largely confirmed the metataxonomics data and indicated significantly higher yeast counts in Center 1 TSHR-immunized mice. We also compared the gut microbiota between immunization groups within Center 2, comprising the TSHR- or βgal control-immunized mice and naïve untreated mice. We observed a shift of the TSHR-immunized mice bacterial communities described by the beta diversity weighted Unifrac. Furthermore, we observed a significant positive correlation between the presence of Firmicutes and orbital-adipogenesis specifically in TSHR-immunized mice.

Conclusions: The significant differences observed in microbiota composition from BALBc mice undergoing the same immunization protocol in comparable specific-pathogen-free (SPF) units in different centers support a role for the gut microbiota in modulating the induced response. The gut microbiota might also contribute to the heterogeneity of induced response since we report potential disease-associated microbial taxonomies and correlation with ocular disease.

Keywords: Firmicutes; Graves’ disease; Graves’ orbitopathy; Gut microbiota; Induced animal model; Metataxonomics; Orbital adipogenesis; TSHR.

Conflict of interest statement

Ethics approval and consent to participate

The study was approved by the North Rhine Westphalian State Agency for Nature, Environment and Consumer Protection, Germany and by the Ethics Committee of King’s College London, United Kingdom (UK).

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Comparative analysis of the gut microbiota in independent animal units. a Box and whisker plot of the alpha diversity indices for richness (Chao1 and observed OTUs indices) and evenness (Shannon index) of the bacterial communities in TSHR-immunized mice housed in Center 1 (blue) and Center 2 (red), respectively. Tukey’s HSD post hoc: Chao1, P = 0.01; observed OTUs, P < 0.001; Shannon, P = 0.08. b Annotated heatmap based on Spearman distance and Ward hierarchical clustering of the top 30 genera shows how well the two locations cluster together. Taxonomy explanation includes genera, family, and phylum, which are entered in order of abundance. Genus abundance is described by the change in the intensity of the gray color, as annotated. c Multidimensional scaling plot (MDS) based on the weighted Unifrac distances between the two animal units. PERMANOVA with 999 permutations P = 0.005. d Differentially abundant family from a pairwise comparison with Welch’s t test with 95% confidence intervals (STAMP). e Box and whisker plot culture results from intestinal scraped samples derived from TSHR-immunized mice from Center 1 and Center 2. Results are expressed as a Log(x + 1) transformed colony-forming units/gram feces (cfu/g). P values: * P < 0.05; ** P < 0.001; *** P < 0.005

Fig. 2

Gut microbiota composition in TSHR-immunized mice and control mice in Center 2 at final timepoint. a Box and whisker plots describing the measurement of alpha diversity (Chao, ACE, and Shannon indices). b Non-metric dimensional scaling (NMDS) plot of weighted Unifrac distances showed a spatial separation of microbial communities according to the immunizations. PERMANOVA based on 999 permutations P = 0.001. c Boxplot of the phylum counts according to immunizations. ANOVA on phylum counts BH adjusted P < 0.0001 and pairwise T test between Bacteroidetes-Firmicutes counts adjusted P = 0.0003. d Non-Metric Dimensional Scaling (NMDS) plot based on weighted Unifrac distances shows spatial separation of the microbial community according to the immunization and caging within Center 2. Mice were co-housed according to their immunization at a maximum of four animals; cages are described by different shapes as in the legend. No significant difference in cage effect is observed. PERMANOVA based on cage effect (999 permutations) for all comparisons P = 0.12. P values: * P ≤ 0.05; ** P = 0.01

Fig. 3

Time-course analysis of GO preclinical fecal microbiota during the immunization protocol. Box and whisker plots of alpha diversity such as Chao (a) and Shannon (b) indices showed differences over time. c Phylum dynamics over time and between immunizations. Firmicutes and Bacteroidetes were the most abundant phyla, showing differences with time and immunizations. Significant differences among timepoints have been observed at the Firmicutes/Bacteroidetes ratio, in particular between the baseline T0 and the last timepoint T4, but not related to immunization. A significant difference in the ratio was observed after 3 weeks from the first injection (T1) between βgal and TSHR. P values: * P ≤ 0.05; ** P = 0.01

Fig. 4

Correlating the gut microbiota and disease features in Center 2 TSHR group. a Spearman correlation coefficient strength (Rho) of phylum counts from TSHR mice in Center 2. Firmicutes and Bacteoridetes showed a strong negative correlation between each other. A positive correlation between the one-genus phylum Deferribacteres and the level of thyroid-stimulating antibodies (TSAb) has been observed. Correlations with P < 0.05 are shown and strength of the Rho coefficient is represented by the change in the color intensity. fT4, free thyroid hormone thyroxine levels; TSAb, thyroid stimulating antibodies; TSBAb, thyroid-stimulating blocking antibodies (as a percentage values). b Enriched Firmicutes genus Intestinimonas between Center 1 (blue) and Center 2 (red) showed a strong negative correlation with the percentage of thyroid-stimulating blocking antibodies (TSBAbs) at 95% confidence interval in Center 1 (Rho = − 0.8, P = 0.04), but not in Center 2

Fig. 5

Correlation of the gut microbiota composition with clinical features and differences in Center 2 mice. a Correlation plot of phyla and the orbital adipogenesis value. Spearman correlation coefficient strength (Rho) as indicated by the colored bar. Firmicutes and Bacteoridetes showed a strong negative correlation between each other. A positive correlation between Firmicutes and a negative correlation with Bacteroidetes OTUs and the adipogenesis value (calculated in the orbit) has been observed. Adipogenesis clustered closer to the Firmicutes and Bacteroidetes value according to the complete linkage method for hierarchical clustering. Only P < 0.05 are shown. b Positive strong correlation of the Firmicutes/Bacteroidetes ratio with the adipogenesis value (calculated in the orbit) resulted significant in TSHR-immunized group but not in the βgal group. c Spearman correlation coefficient (Rho) of genera among phyla Bacteroidetes and Firmicutes and the orbital adipogenesis values. The strength of the correlation coefficient is represented on x-axis: bars on the left represent a negative correlation coefficient, while bars on the right represent a positive correlation coefficient. Correlations with P < 0.05 are shown; order of entrance depends on their P values: * P < 0.05; ** P < 0.1; *** P < 0.005. d Spearman correlation coefficient plot of the Box-Cox transformed microbiological counts and disease features in Center 2 TSHR-immunized mice. Feature clustering was according to the complete linkage method for hierarchical clustering. Only correlations with P < 0.05 are shown and strength of the correlation coefficient is represented by the change in the color intensity. fT4, free thyroid hormone thyroxine levels; TSAb, thyroid-stimulating antibodies; TSBAb, thyroid-stimulating blocking antibodies (as a percentage values)