Modulating gut microbiota in a mouse model of Graves’ orbitopathy and its impact on induced disease

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.

The Role of the Microbiota in Graves’ Disease and Graves’ Orbitopathy

“…To investigate whether the above-observed correlation indicates causation, the same research team altered the microbiota composition before the TSHR immunization by antibiotic vancomycin, probiotic Lab4, and fecal material transfer (FMT) from GO patients ( Moshkelgosha et al., 2021 ). The antibiotic vancomycin was administered through drinking water, and probiotic Lab4 and FMT powder were administered through gavage.…”

“…Lab4 exacerbated TSHR-induced autoimmune GD and GO-like phenotypes. Thus, although Lab4 increases orbital Tregs in normal mice, it cannot prevent TSHR-induced tolerance breakdown ( Moshkelgosha et al., 2021 ).…”

“…In summary ( Table 1 and Figure 1 ), experimental GD/GO models of the same mouse strain but housed in two different animal facilities ( Masetti et al., 2018 ), or different mouse strains from the same animal facility ( Moshkelgosha et al., 2018 ), revealed significant differences in gut microbiota composition which explain variations in clinical manifestations. Modulating gut microbiota can change the incidence and severity of GD/GO ( Su et al., 2020 ; Moshkelgosha et al., 2021 ). These results uncovered a crucial role of gut microbiota in initiating and developing GD/GO mouse models ( Masetti and Ludgate, 2020 ).…”

“…The TSHR plasmid-immunized GD/GO mice in Essen also had more abundant Lactobacillaceae ( Figure 1 ) ( Berchner-Pfannschmidt et al., 2016 ; Masetti et al., 2018 ). Lactobacillus is generally not pathogenic in the human body, and it is a recognized element in some probiotics such as Lab4 ( Moshkelgosha et al., 2021 ). However, some studies also suggested that Lactobacillus can induce macrophages to secrete proinflammation cytokines, such as IL-6 and TNF-α ( Rocha-Ramírez et al., 2017 ), and the abundance of Lactobacillus increased in autoimmune hepatitis ( Wei et al., 2020 ) and Crohn’s disease ( Wang et al., 2014 ; Effenberger et al., 2021 ).…”

“…These results are consistent with the observed effects of Lab4 on mice. Lab4 increases orbital Tregs in normal mice but exacerbates TSHR-induced autoimmune GD and GO-like phenotypes ( Figure 1 ) ( Moshkelgosha et al., 2021 ).…”