Sajad Moshkelgosha1,2, Hedda Luise Verhasselt3,4, Giulia Masetti5,6,7, Anja Eckstein8, Marian Ludgate9, Utta Berchner-Pfannschmidt10, Danila Covelli4,11, Filippo Biscarini6,12, Mareike Horstmann1, Anke Daser13, Astrid M Westendorf3, Christoph Jesenek1, Svenja Philipp1, Salvador Diaz-Cano14, J Paul Banga1, Daryn Michael4, Sue Plummer4, Julian R Marchesi15,16. 1. Molecular Ophthalmology, Department of Ophthalmology, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany. 2. Current address: Latner Thoracic Surgery Laboratories, Toronto General Research Institute, University Health Network and University of Toronto, Toronto, Canada. 3. Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. 4. Cultech Ltd., Baglan, Port Talbot, UK. 5. Division of Infection & Immunity, School of Medicine, Cardiff University, UHW main building, Heath Park, Cardiff, CF14 4XW, UK. 6. Department of Bioinformatics, PTP Science Park Srl, Lodi, Italy. 7. Current address: Computational metagenomics, Department CIBIO, University of Trento, Trento, Italy. 8. Department of Ophthalmology, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany. Anja.Eckstein@uk-essen.de. 9. Division of Infection & Immunity, School of Medicine, Cardiff University, UHW main building, Heath Park, Cardiff, CF14 4XW, UK. Ludgate@cardiff.ac.uk. 10. Molecular Ophthalmology, Department of Ophthalmology, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany. Utta.Berchner-Pfannschmidt@uk-essen.de. 11. Graves' Orbitopathy Center, Endocrinology, Department of Clinical Sciences and Community Health, Fondazione Ca'Granda IRCCS, University of Milan, Milan, Italy. 12. Italian National Research Council (CNR), Milano, Italy. 13. Department of Ophthalmology, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany. 14. Department of Histopathology, King's College Hospital, King's College, London, UK. 15. School of Biosciences, Cardiff University, Cardiff, UK. 16. Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK.
Abstract
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. Video abstract.
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 withTSHR-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 withTSHR-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-TSHRmice, 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 withAkkermansiaspp. 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 TSHRmice. 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. Video abstract.
Entities:
Keywords:
Graves’ disease; Graves’ orbitopathy; Gut microbiota; Human fecal microbiota transplant; Microbiome modulation; Murine model; Probiotics; Vancomycin
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