Gut microbiota in experimental murine model of Graves' orbitopathy established in different environments may modulate clinical presentation of disease
Giulia Masetti, Sajad Moshkelgosha, Hedda-Luise Köhling, Danila Covelli, Jasvinder Paul Banga, Utta Berchner-Pfannschmidt, Mareike Horstmann, Salvador Diaz-Cano, Gina-Eva Goertz, Sue Plummer, Anja Eckstein, Marian Ludgate, Filippo Biscarini, Julian Roberto Marchesi, INDIGO consortium, Giulia Masetti, Sajad Moshkelgosha, Hedda-Luise Köhling, Danila Covelli, Jasvinder Paul Banga, Utta Berchner-Pfannschmidt, Mareike Horstmann, Salvador Diaz-Cano, Gina-Eva Goertz, Sue Plummer, Anja Eckstein, Marian Ludgate, Filippo Biscarini, Julian Roberto Marchesi, INDIGO consortium
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 participateThe 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 interestsThe authors declare that they have no competing interests.
Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References
- Ericsson AC, Davis JW, Spollen W, Bivens N, Givan S, et al. Effects of vendor and genetic background on the composition of the fecal microbiota of inbred mice. PLoS One. 2015;10:e0116704. doi: 10.1371/journal.pone.0116704.
- Hufeldt MR, Nielsen DS, Vogensen FK, Midtvedt T, Hansen AK. Variation in the gut microbiota of laboratory mice is related to both genetic and environmental factors. Compar Med. 2010;60:336–347.
- Draman M, Ludgate M. Thyroid eye disease—an update. Exp Rev Ophthalmol. 2016;11:1–12. doi: 10.1080/17469899.2016.1202113.
- McLachlan SM, Rapoport B. Breaking tolerance to thyroid antigens: changing concepts in thyroid autoimmunity. Endocr Rev. 2014;35:59–105. doi: 10.1210/er.2013-1055.
- Morshed SA, Davies TF. Graves’ disease mechanisms: the role of stimulating, blocking and cleavage region TSHR receptor antibodies. Horm Metab Res. 2015;47(Suppl 10):727–734.
- Bahn RS. Graves’ ophthalmopathy. New Engl J Med. 2010;362:726–738. doi: 10.1056/NEJMra0905750.
- Banga JP, Moshkelgosha S, Berchner-Pfannschmidt U, Eckstein A. Modelling Graves’ orbitopathy in experimental Graves’ disease. Horm Metab Res. 2015; doi: 10.1055/s-0035-1555956.
- Ludgate M. Animal models of Graves’ disease. Eur J Endocrinol. 2000;142:1–8. doi: 10.1530/eje.0.1420001.
- Many MC, Costagliola S, Detrait M, Denef JF, Vassart G, Ludgate M. Development of an animal model of autoimmune thyroid eye disease. J Immunol. 1999;162:4966–4974.
- Baker G, Mazziotti G, von Ruhland C, Ludgate M. Reevaluating thyrotropin receptor-induced mouse models of Graves’ disease and ophthalmopathy. Endocrinology. 2005;146:835–844. doi: 10.1210/en.2004-1015.
- Bhattacharyya KK, Coenen MJ, Bahn RS. Effect of environmental pathogens on the TSHR-directed immune response in an animal model of Graves’ disease. Thyroid 2005;15:422–6.
- Berchner-Pfannschmidt U, Moshkelgosha S, Diaz-Cano S, Edelmann B, Görtz G-EE, Horstmann M, et al. 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. Endocrinology. 2016;157:1673–82.
- Moshkelgosha S, So P-W, Deasy N, Diaz-Cano S, Banga J. Retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves’ orbitopathy induced by thyrotropin receptor plasmid-in vivo electroporation. Endocrinology. 2013;154:3008–3015. doi: 10.1210/en.2013-1576.
- Murri M, Leiva I, Gomez-Zumaquero JM, Tinahones FJ, Cardona F, et al. Gut microbiota in children with type 1 diabetes differs from that in healthy children: a case-control study. BMC Med. 2013;11:46. doi: 10.1186/1741-7015-11-46.
- Brown CT, Davis-Richardson AG, Giongo A, Gano KA, Crabb DB, et al. Gut microbiome metagenomics analysis suggests a functional model for the development of autoimmunity for type 1 diabetes. PLoS One. 2011;6:25792. doi: 10.1371/journal.pone.0025792.
- Frank DN, St. Amand AL, Feldman RA, Boedeker EC, Harpaz N, Pace NR. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel disease. Proc Natl Acad Sci U S A. 2007;104:13780–13785. doi: 10.1073/pnas.0706625104.
- Scanlan PD, Shanahan F, O’Mahony C, Marchesi JR. Culture-independent analyses of temporal variation of the dominant fecal microbiota and targeted bacterial subgroups in Crohn’s disease. J Clin Microbiol. 2006;44:3980–3988. doi: 10.1128/JCM.00312-06.
- Laukens D, Brinkman BM, Raes J, De Vos M, Vandenabeele P. Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design. FEMS Microbiol Rev. 2016;40:117–132. doi: 10.1093/femsre/fuv036.
- Ochoa-Repáraz J, Mielcarz DW, Ditrio LE, Burroughs AR, Foureau DM, et al. Role of gut commensal microflora in the development of experimental autoimmune encephalomyelitis. J Immunol. 2009;183:6041–6050. doi: 10.4049/jimmunol.0900747.
- Lee YK, Menezes JS, Umesaki Y, Mazmanian SK. Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S A. 2011;108(Suppl 1):4615–4622. doi: 10.1073/pnas.1000082107.
- Covelli D, Ludgate M. The thyroid, the eyes and the gut: a possible connection. J Endocrinol Investig. 2017; 10.1007/s40618-016-0594-6.
- Zhao SX, Tsui S, Cheung A, Douglas RS, Smith TJ, Banga JP. Orbital fibrosis in a mouse model of graves’ disease induced by genetic immunization of thyrotropin receptor cDNA. J Endocrinol. 2011;210:369–377. doi: 10.1530/JOE-11-0162.
- Box GEP, Cox DR. An analysis of transformations. J R Stat Soc B. 1964;26:211–243.
- Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009; 10.1128/AEM.01541-09.
- Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011;27:2194–2200. doi: 10.1093/bioinformatics/btr381.
- Cole JR, Wang Q, Cardenas E, Fish J, Chai B, et al. The ribosomal database project: improved alignments and new tools for rRNA analysis. Nucl Acids Res. 2009;37:D141–D145. doi: 10.1093/nar/gkn879.
- Price MN, Dehal PS, Arkin AP. FastTree 2–approximately maximum-likelihood trees for large alignments. PLoS One. 2010;5:e9490. doi: 10.1371/journal.pone.0009490.
- Parks DH, Tyson GW, Hugenholtz P, Beiko RG. STAMP: statistical analysis of taxonomic and functional profiles. Bioinformatics. 2014; 10.1093/bioinformatics/btu494.
- Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R. UniFrac: an effective distance metric for microbial community comparison. ISME J. 2011;5:169–172. doi: 10.1038/ismej.2010.133.
- Anderson MJ. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 2001;26:32–46.
- Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 10.1093/bioinformatics/btp616.
- Köhling HL, Plummer SF, Marchesi JR, Davidge KS, Ludgate M. The microbiota and autoimmunity: their role in thyroid autoimmune diseases. Clin Immunol. 2017; 10.1016/j.clim.2017.07.001.
- Chao A, et al. Species estimation and application. In: Kotz S, Balakishnan N, et al., editors. Encyclopedia of statistical sciences. New York: Wiley; 2005. pp. 7907–7915.
- Mariat D, Firmesse O, Levenez F, Guimarăes VD, Sokol H, et al. The firmicutes/bacteroidetes ratio of the human microbiota changes with age. BMC Microbiol. 2009;9:123. doi: 10.1186/1471-2180-9-123.
- Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature. 2006;444:1022–1023. doi: 10.1038/4441022a.
- Vecchiatti S, Guzzo M, Caldini E, Bisi H, Longatto-Filho A, et al. Iodine increases and predicts incidence of thyroiditis in NOD mice: histopathological and ultrastructural study. Exp and therap med. 2013;5:603–607. doi: 10.3892/etm.2012.826.
- Rapoport B, Aliesky HA, Banuelos B, Chen C-RR, McLachlan SM. A unique mouse strain that develops spontaneous, iodine-accelerated, pathogenic antibodies to the human thyrotrophin receptor. J Immunol. 2015;194:4154–4161. doi: 10.4049/jimmunol.1500126.
- Jakobsson H, Rodríguez-Piñeiro A, Schütte A, Ermund A, Boysen P, et al. The composition of the gut microbiota shapes the colon mucus barrier. EMBO Rep. 2015;16:164–177. doi: 10.15252/embr.201439263.
- Kläring K, Hanske L, TPN B, Charrier C, Blaut M, et al. Intestinimonas butyriciproducens gen. nov., sp. nov., a butyrate-producing bacterium from the mouse intestine. Int J Syst Evol Microbiol. 2013; 10.1099/ijs.0.051441-0.
- Bui TPN, Ritari J, Boeren S, de Waard P, Plugge CM, Vos WM. Production of butyrate from lysine and the Amadori product fructoselysine by a human gut commensal. Nat Commun. 2015;6:10062. doi: 10.1038/ncomms10062.
- Million M, Lagier JC, Yahav D, Paul M. Gut bacterial microbiota and obesity. Clin Microb Inf. 2013;19:305–313. doi: 10.1111/1469-0691.12172.
- Anderson DJ, Axel R. Molecular probes for the development and plasticity of neural crest derivatives. Cell. 1985;42(2):649–662. doi: 10.1016/0092-8674(85)90122-9.
- McCafferty J, Mühlbauer M, Gharaibeh R, Arthur J, Perez-Chanona E, et al. Stochastic changes over time and not founder effects drive cage effects in microbial community assembly in a mouse model. ISME J. 2013;7:2116–2125. doi: 10.1038/ismej.2013.106.
- Juers DH, Matthews BW, Huber RE. LacZ galactosidase: structure and function of an enzyme of historical and molecular biological importance. Protein Sci. 2012;21:1792–1807. doi: 10.1002/pro.2165.
- Kaneda T, Honda A, Hakozaki A, Fuse T, Muto A, Yoshida T. An improved graves’ disease model established by using in vivo electroporation exhibited long-term immunity to hyperthyroidism in BALB/c mice. Endocrinology. 2007;148:2335–2344. doi: 10.1210/en.2006-1077.
- Wu H-J, Ivanov I, Darce J, Hattori K, Shima T, et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity. 2010;32:815–827. doi: 10.1016/j.immuni.2010.06.001.
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