Gut Microbiome in BALB/c and C57BL/6J Mice Undergoing Experimental Thyroid Autoimmunity Associate with Differences in Immunological Responses and Thyroid Function

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.

Key words

Graves’ disease - autoimmunity - TSH-receptor antibodies

^* Shared first authorship

• References

• 1 Antonelli A, Ferrari SM, Corrado A, Di Domenicantonio A, Fallahi P. Autoimmune thyroid disorders. Autoimmun Rev 2015; 14: 174-180
• 2 Weetman AP. Graves' disease. N Eng J Med 2000; 343: 1236-1248
• 3 Rees Smith B, McLachlan SM, Furmaniak J. Autoantibodies to the thyrotropin receptor. Endocr Rev 1988; 9: 106-121
• 4 Davies TF, Ando T, Lin RY, Tomer Y, Latif R. Thyrotropin receptor-associated diseases: From adenomata to Graves disease. J Clin Invest 2005; 115: 1972-1983
• 5 Bahn RS. Graves' ophthalmopathy. N Eng J Med 2010; 362: 726-738
• 6 Tomer Y. Mechanisms of autoimmune thyroid diseases: From genetics to epigenetics. Ann Rev Pathol 2014; 9: 147-156
• 7 Hargreaves CE, Grasso M, Hampe CS, Stenkova A, Atkinson S, Joshua GW, Wren BW, Buckle AM, Dunn-Walters D, Banga JP. Yersinia enterocolitica provides the link between thyroid-stimulating antibodies and their germline counterparts in Graves' disease. J Immunol 2013; 190: 5373-5381
• 8 Kohling HL, Plummer SF, Marchesi JR, Davidge KS, Ludgate M. The microbiota and autoimmunity: Their role in thyroid autoimmune diseases. Clin Immunol 2017; 183: 63-74
• 9 Covelli D, Ludgate M. The thyroid, the eyes and the gut: A possible connection. J Endocrinol Invest 2017; 40: 567-576
• 10 Rapoport B, Aliesky HA, Banuelos B, Chen CR, McLachlan SM. A unique mouse strain that develops spontaneous, iodine-accelerated, pathogenic antibodies to the human thyrotrophin receptor. J Immunol 2015; 194: 4154-4161
• 11 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
• 12 Wiesweg B, Johnson KT, Eckstein AK, Berchner-Pfannschmidt U. Current insights into animal models of Graves' disease and orbitopathy. Horm Metab Res 2013; 45: 549-555
• 13 Nagayama Y, Kita-Furuyama M, Ando T, Nakao K, Mizuguchi H, Hayakawa T, Eguchi K, Niwa M. A novel murine model of Graves' hyperthyroidism with intramuscular injection of adenovirus expressing the thyrotropin receptor. J Immunol 2002; 168: 2789-2794
• 14 Chen CR, Pichurin P, Nagayama Y, Latrofa F, Rapoport B, McLachlan SM. The thyrotropin receptor autoantigen in Graves disease is the culprit as well as the victim. J Clin Invest 2003; 111: 1897-1904
• 15 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
• 16 Gilbert JA, Gianoukakis AG, Salehi S, Moorhead J, Rao PV, Khan MZ, McGregor AM, Smith TJ, Banga JP. Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways. J Immunol 2006; 176: 5084-5092
• 17 Chen CR, Aliesky H, Pichurin PN, Nagayama Y, McLachlan SM, Rapoport B. Susceptibility rather than resistance to hyperthyroidism is dominant in a thyrotropin receptor adenovirus-induced animal model of Graves' disease as revealed by BALB/c-C57BL/6 hybrid mice. Endocrinology 2004; 145: 4927-4933
• 18 Johnson KT, Wiesweg B, Schott M, Ehlers M, Muller M, Minich WB, Nagayama Y, Gulbins E, Eckstein AK, Berchner-Pfannschmidt U. Examination of orbital tissues in murine models of Graves' disease reveals expression of UCP-1 and the TSHR in retrobulbar adipose tissues. Horm Metab Res 2013; 45: 401-407
• 19 Nagayama Y, McLachlan SM, Rapoport B, Niwa M. A major role for non-major histocompatibility complex genes but not for microorganisms in a novel murine model of Graves' hyperthyroidism. Thyroid 2003; 13: 233-238
• 20 Seetharamaiah GS, Land KJ. Differential Susceptibility of BALB/c and BALB/cBy mice to Graves' hyperthyroidism. Thyroid 2006; 16: 651-658
• 21 Berchner-Pfannschmidt U, Moshkelgosha S, Diaz-Cano S, Edelmann B, Gortz GE, Horstmann M, Noble A, Hansen W, Eckstein A, Banga JP. Comparative Assessment of Female Mouse Model of Graves' Orbitopathy Under Different Environments, Accompanied by Proinflammatory Cytokine and T-Cell Responses to Thyrotropin Hormone Receptor Antigen. Endocrinology 2016; 157: 1673-1682
• 22 Moshkelgosha S, So PW, Deasy N, Diaz-Cano S, Banga JP. Cutting Edge: 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
• 23 Ungerer M, Fassbender J, Li Z, Munch G, Holthoff HP. Review of Mouse Models of Graves' Disease and Orbitopathy-Novel Treatment by Induction of Tolerance. Clin Rev Allergy Immunol 2017; 52: 182-193
• 24 Masetti G, Moshkelgosha S, Kohling HL, Covelli D, Banga JP, Berchner-Pfannschmidt U, Horstmann M, Diaz-Cano S, Goertz GE, Plummer S, Eckstein A, Ludgate M, Biscarini F, Marchesi JR. Consortium I . Gut microbiota in experimental murine model of Graves' orbitopathy established in different environments may modulate clinical presentation of disease. Microbiome 2018; 6: 97
• 25 Banga JPMS, Berchner-Pfannschmidt U, Eckstein A. An animal model of Graves' orbitopathy in 'Animal models of ophthalmic diseases'. Chan CC. (ed) Berlin: Springer; 2017: 117-124
• 26 Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 2009; 75: 7537-7541
• 27 Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM. The Ribosomal Database Project: Improved alignments and new tools for rRNA analysis. Nucleic Acids Res 2009; 37: D141-D145
• 28 Lozupone C, Knight R. UniFrac: A new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 2005; 71: 8228-8235
• 29 Ericsson AC, Davis JW, Spollen W, Bivens N, Givan S, Hagan CE, McIntosh M, Franklin CL. Effects of vendor and genetic background on the composition of the fecal microbiota of inbred mice. PLoS One 2015; 10: e0116704
• 30 Friswell MK, Gika H, Stratford IJ, Theodoridis G, Telfer B, Wilson ID, McBain AJ. Site and strain-specific variation in gut microbiota profiles and metabolism in experimental mice. PLoS One 2010; 5: e8584
• 31 Schluter A, Horstmann M, Diaz-Cano S, Plohn S, Stahr K, Mattheis S, Oeverhaus M, Lang S, Flogel U, Berchner-Pfannschmidt U, Eckstein A, Banga JP. Genetic immunization with mouse thyrotrophin hormone receptor plasmid breaks self-tolerance for a murine model of autoimmune thyroid disease and Graves' orbitopathy. Clin Exp Immunol 2018; 191: 255-267
• 32 Hashimoto T, Perlot T, Rehman A, Trichereau J, Ishiguro H, Paolino M, Sigl V, Hanada T, Hanada R, Lipinski S, Wild B, Camargo SM, Singer D, Richter A, Kuba K, Fukamizu A, Schreiber S, Clevers H, Verrey F, Rosenstiel P, Penninger JM. ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation. Nature 2012; 487: 477-481
• 33 Correa-Oliveira R, Fachi JL, Vieira A, Sato FT, Vinolo MA. Regulation of immune cell function by short-chain fatty acids. Clin Transl Immunol 2016; 5: e73
• 34 Marino E, Richards JL, McLeod KH, Stanley D, Yap YA, Knight J, McKenzie C, Kranich J, Oliveira AC, Rossello FJ, Krishnamurthy B, Nefzger CM, Macia L, Thorburn A, Baxter AG, Morahan G, Wong LH, Polo JM, Moore RJ, Lockett TJ, Clarke JM, Topping DL, Harrison LC, Mackay CR. Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes. Nat Immunol 2017; 18: 552-562
• 35 Jahnsen FL, Baekkevold ES, Hov JR, Landsverk OJ. Do Long-Lived Plasma Cells Maintain a Healthy Microbiota in the Gut?. Trends Immunol 2018; 39: 196-208
• 36 Kim M, Qie Y, Park J, Kim CH. Gut Microbial Metabolites Fuel Host Antibody Responses. Cell Host Microbe 2016; 20: 202-214
• 37 Baron EJ. Bilophila wadsworthia: A unique Gram-negative anaerobic rod. Anaerobe 1997; 3: 83-86
• 38 Cotta MA, Whitehead TR, Falsen E, Moore E, Lawson PA. Robinsoniella peoriensis gen. nov., sp. nov., isolated from a swine-manure storage pit and a human clinical source. Int J Syst Evol Microbiol 2009; 59: 150-155
• 39 Karlsson H, Larsson P, Wold AE, Rudin A. Pattern of cytokine responses to gram-positive and gram-negative commensal bacteria is profoundly changed when monocytes differentiate into dendritic cells. Infect Immun 2004; 72: 2671-2678
• 40 Skovbjerg S, Martner A, Hynsjo L, Hessle C, Olsen I, Dewhirst FE, Tham W, Wold AE. Gram-positive and gram-negative bacteria induce different patterns of cytokine production in human mononuclear cells irrespective of taxonomic relatedness. J Interferon Cytokine Res 2010; 30: 23-32
• 41 Zhao F, Feng J, Li J, Zhao L, Liu Y, Chen H, Jin Y, Zhu B, Wei Y. Alterations of the Gut Microbiota in Hashimoto's Thyroiditis Patients. Thyroid 2018; 28: 175-186
• 42 Cekanaviciute E, Yoo BB, Runia TF, Debelius JW, Singh S, Nelson CA, Kanner R, Bencosme Y, Lee YK, Hauser SL, Crabtree-Hartman E, Sand IK, Gacias M, Zhu Y, Casaccia P, Cree BAC, Knight R, Mazmanian SK, Baranzini SE. Gut bacteria from multiple sclerosis patients modulate human T cells and exacerbate symptoms in mouse models. Proc Natl Acad Sci U S A 2017; 114: 10713-10718
• 43 Wekerle H. Brain Autoimmunity and Intestinal Microbiota: 100 Trillion Game Changers. Trends Immunol 2017; 38: 483-497
• 44 Paun A, Yau C, Danska JS. The Influence of the Microbiome on Type 1 Diabetes. J Immunol 2017; 198: 590-595
• 45 Wen L, Ley RE, Volchkov PY, Stranges PB, Avanesyan L, Stonebraker AC, Hu C, Wong FS, Szot GL, Bluestone JA, Gordon JI, Chervonsky AV. Innate immunity and intestinal microbiota in the development of Type 1 diabetes. Nature 2008; 455: 1109-1113
• 46 De Riva A, Wallberg M, Ronchi F, Coulson R, Sage A, Thorne L, Goodfellow I, McCoy KD, Azuma M, Cooke A, Busch R. Regulation of type 1 diabetes development and B-cell activation in nonobese diabetic mice by early life exposure to a diabetogenic environment. PloS One 2017; 12: e0181964
• 47 Zhou JS, Gill HS. Immunostimulatory probiotic Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 do not induce pathological inflammation in mouse model of experimental autoimmune thyroiditis. Int J Food Microbiol 2005; 103: 97-104
• 48 Virili C, Centanni M. Does microbiota composition affect thyroid homeostasis?. Endocrine 2015; 49: 583-587
• 49 Horai R, Zarate-Blades CR, Dillenburg-Pilla P, Chen J, Kielczewski JL, Silver PB, Jittayasothorn Y, Chan CC, Yamane H, Honda K, Caspi RR. Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Site. Immunity 2015; 43: 343-353
• 50 Zarate-Blades CR, Horai R, Mattapallil MJ, Ajami NJ, Wong M, Petrosino JF, Itoh K, Chan CC, Caspi RR. Gut microbiota as a source of a surrogate antigen that triggers autoimmunity in an immune privileged site. Gut Microbes 2017; 8: 59-66