Macrophage-Orbital Fibroblast Interaction and Hypoxia Promote Inflammation and Adipogenesis in Graves' Orbitopathy.

The inflammatory eye disease Graves' orbitopathy (GO) is the main complication of autoimmune Graves' disease. In previous studies we have shown that hypoxia plays an important role for progression of GO. Hypoxia can maintain inflammation by attracting inflammatory cells such as macrophages (MQ). Herein, we investigated the interaction of MQ and orbital fibroblasts (OF) in context of inflammation and hypoxia. We detected elevated levels of the hypoxia marker HIF-1α, the MQ marker CD68, and inflammatory cytokines TNFα, CCL2, CCL5, and CCL20 in GO biopsies. Hypoxia stimulated GO tissues to release TNFα, CCL2, and CCL20 as measured by multiplex enzyme-linked immunosorbent assay (ELISA). Further, TNFα and hypoxia stimulated the expression of HIF-1α, CCL2, CCL5, and CCL20 in OF derived from GO tissues. Immunofluorescence confirmed that TNFα-positive MQ were present in the GO tissues. Thus, interaction of M1-MQ with OF under hypoxia also induced HIF-1α, CCL2, and CCL20 in OF. Inflammatory inhibitors etanercept or dexamethasone prevented the induction of HIF-1α and release of CCL2 and CCL20. Moreover, co-culture of M1-MQ/OF under hypoxia enhanced adipogenic differentiation and adiponectin secretion. Dexamethasone and HIF-1α inhibitor PX-478 reduced this effect. Our findings indicate that GO fat tissues are characterized by an inflammatory and hypoxic milieu where TNFα-positive MQ are present. Hypoxia and interaction of M1-MQ with OF led to enhanced secretion of chemokines, elevated hypoxic signaling, and adipogenesis. In consequence, M1-MQ/OF interaction results in constant inflammation and tissue remodeling. A combination of anti-inflammatory treatment and HIF-1α reduction could be an effective treatment option.

An Early Wave of Macrophage Infiltration Intertwined with Antigen-Specific Proinflammatory T Cells and Browning of Adipose Tissue Characterizes the Onset of Orbital Inflammation in a Mouse Model of Graves' Orbitopathy.

Background: Graves' orbitopathy (GO) is an autoimmune-driven manifestation of Graves' disease (GD) where pathogenic autoantibodies to the thyrotropin receptor (TSHR) activate orbital fibroblasts/preadipocytes in the orbital tissue to induce inflammation and extracellular matrix deposition. Since there are significant limitations to study immunological and proinflammatory mediator expression in early and during disease progression in GO patients, we used our experimental mouse model to elucidate early pathogenic processes. Methods: We have developed a robust mouse model of GD/GO induced by electroporation immunization of plasmid encoding human TSHR A-subunit, comprising multiple injections over a course of 15 weeks to fully recapitulate the orbital pathology. In this study, we investigated kinetics of GO development in the model by serial analyses of immunological and cellular parameters during course of orbital inflammation. Results: Pathogenic anti-TSHR antibodies with thyroid-stimulating properties developed early after the second immunization step with concomitant induction of hyperthyroidism. Examination of orbital tissue showed an early wave of macrophage infiltration followed subsequently by CD3+ T cells into the orbital tissue. Examination of antigen-specific T cell activity using recombinant human A-subunit protein showed high CD8+ T cell proliferation during this early phase of disease onset, whereas effector CD4+ T cells and CD25+FOXP3+ regulatory T cells (Tregs) were downregulated. The early phase of disease was also characterized by abundant presence of proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Moreover, as the disease progressed, there was significant increase in browning of orbital fat tissue, which may be dependent on the proinflammatory milieu and/or the increased thyroid hormone levels during the established hyperthyroid status. Conclusions: This work revealed early infiltration of macrophages in the orbital region and induction of pathogenic anti-TSHR antibodies during disease onset in the model. This was followed subsequently by influx of CD8+ T cells specific for TSHR coupled with reduction in Tregs and substantial increase in brown adipose tissue. These new insights into the development of orbital inflammation in the model have implications for testing new therapeutic regimens by targeting macrophage function during early phases of orbital inflammation in the model.

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

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 (ßgal; 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-ßgal; 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.

Pathogenic Phenotype of Adipogenesis and Hyaluronan in Orbital Fibroblasts From Female Graves' Orbitopathy Mouse Model.

A mouse model of Graves' orbitopathy (GO) induced by genetic immunization of human TSH receptor (TSHR) A-subunit encoding plasmid has recently been established. The orbital pathology was characterized by adipogenesis, myopathy and fibrosis. Human orbital fibroblasts (OFs) express TSHR and IGF-1 receptor (IGF-1R) and are considered to be pathogenic in GO. We established conditions for growing ex vivo cultures of mouse OFs (mOFs) from orbital tissue of animals undergoing GO and controls. Early passage mOFs showed characteristic fibroblast morphology and expressed mesenchymal stem cell markers including a strong expression of CD90.2 and CD40, whereas display of CD73 and all other leucocyte markers was uniformly absent. Importantly, OFs derived from GO mice expressed elevated levels of TSHR and IGF-1R and showed enhanced adipogensis compared with controls. Activation of TSHR in mOFs from GO animals with TSH, monoclonal thyroid-stimulating antibody M22, or stimulation of IGF-1R with IGF-1-induced hyaluronan secretion to significantly elevated levels compared with control animals. Hyaluronan synthase 2 was more abundant in OFs derived from GO mice. In conclusion, mOFs established from GO model recapitulate the pathogenicity of human OFs from GO patients by their increased propensity for adipogenesis and hyaluronan production leading to disease activity. To our knowledge, this is the first report to show mOFs from the preclinical GO model have pathogenic properties that will aid in understanding the molecular and genetic changes during progression to adipogenesis and hyaluronan deposition to provide new insights into GO pathogenesis.