Modulating gut microbiota in a mouse model of Graves’ orbitopathy and its impact on induced disease
Creators: name: Sajad Moshkelgosha
name: Hedda Luise Verhasselt, 
name: Masetti, Giulia
name: Covelli, Danila
name: Biscarini, Filippo
name: Horstmann, Mareike
name: Daser, Anke
name: Westendorf, Astrid Maria
name: Jesenek, Christoph
name: Philipp, Svenja
name: Diaz-Cano, Salvador J.
name: J. Paul Banga
name: Daryn Michael
name: Plummer, Sue
name: Marchesi, Julian Roberto
name: Eckstein, Anja
name: Ludgate, Marian
name: Berchner-Pfannschmidt, Utta
name: INDIGO Consortium
Publisher: name: figshare
Year: 2021
Subjects: subject: 110703 Autoimmunity
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: 110704 Cellular Immunology
subject: 110303 Clinical Microbiology
subject: 110306 Endocrinology
subject: 110899 Medical Microbiology not elsewhere classified
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 111301 Ophthalmology
subject: Pathogenesis
subject: 110316 Pathology (excl. Oral Pathology)

description: Background Graves’ disease (GD) is an autoimmune condition in which autoantibodies to the thyrotropin receptor (TSHR) cause hyperthyroidism. About 50% of GD patients also have Graves’ orbitopathy (GO), an intractable disease in which expansion of the orbital contents causes diplopia, proptosis and even blindness. Murine models of GD/GO, developed in different centres, demonstrated significant variation in gut microbiota composition which correlated with TSHR-induced disease heterogeneity. To investigate whether correlation indicates causation, we modified the gut microbiota to determine whether it has a role in thyroid autoimmunity. Female BALB/c mice were treated with either vancomycin, probiotic bacteria, human fecal material transfer (hFMT) from patients with severe GO or ddH2O from birth to immunization with TSHR-A subunit or beta-galactosidase (βgal; age ~ 6 weeks). Incidence and severity of GD (TSHR autoantibodies, thyroid histology, thyroxine level) and GO (orbital fat and muscle histology), lymphocyte phenotype, cytokine profile and gut microbiota were analysed at sacrifice (~ 22 weeks). Results In ddH2O-TSHR mice, 84% had pathological autoantibodies, 67% elevated thyroxine, 77% hyperplastic thyroids and 70% orbital pathology. Firmicutes were increased, and Bacteroidetes reduced relative to ddH2O-βgal; CCL5 was increased. The random forest algorithm at the genus level predicted vancomycin treatment with 100% accuracy but 74% and 70% for hFMT and probiotic, respectively. Vancomycin significantly reduced gut microbiota richness and diversity compared with all other groups; the incidence and severity of both GD and GO also decreased; reduced orbital pathology correlated positively with Akkermansia spp. whilst IL-4 levels increased. Mice receiving hFMT initially inherited their GO donors’ microbiota, and the severity of induced GD increased, as did the orbital brown adipose tissue volume in TSHR mice. Furthermore, genus Bacteroides, which is reduced in GD patients, was significantly increased by vancomycin but reduced in hFMT-treated mice. Probiotic treatment significantly increased CD25⁺ Treg cells in orbital draining lymph nodes but exacerbated induced autoimmune hyperthyroidism and GO. Conclusions These results strongly support a role for the gut microbiota in TSHR-induced disease. Whilst changes to the gut microbiota have a profound effect on quantifiable GD endocrine and immune factors, the impact on GO cellular changes is more nuanced. The findings have translational potential for novel, improved treatments.