Graves' orbitopathy (GO) is a complication in Graves' disease (GD) but mechanistic insights into pathogenesis remain unresolved, hampered by lack of animal model. The TSH receptor (TSHR) and perhaps IGF-1 receptor (IGF-1R) are considered relevant antigens. We show that genetic immunization of human TSHR (hTSHR) A-subunit plasmid leads to extensive remodeling of orbital tissue, recapitulating GO. Female BALB/c mice immunized with hTSHR A-subunit or control plasmids by in vivo muscle electroporation were evaluated for orbital remodeling by histopathology and magnetic resonance imaging (MRI). Antibodies to TSHR and IGF-1R were present in animals challenged with hTSHR A-subunit plasmid, with predominantly TSH blocking antibodies and were profoundly hypothyroid. Orbital pathology was characterized by interstitial inflammation of extraocular muscles with CD3+ T cells, F4/80+ macrophages, and mast cells, accompanied by glycosaminoglycan deposition with resultant separation of individual muscle fibers. Some animals showed heterogeneity in orbital pathology with 1) large infiltrate surrounding the optic nerve or 2) extensive adipogenesis with expansion of retrobulbar adipose tissue. A striking finding that underpins the new model were the in vivo MRI scans of mouse orbital region that provided clear and quantifiable evidence of orbital muscle hypertrophy with protrusion (proptosis) of the eye. Additionally, eyelid manifestations of chemosis, including dilated and congested orbital blood vessels, were visually apparent. Immunization with control plasmids failed to show any orbital pathology. Overall, these findings support TSHR as the pathogenic antigen in GO. Development of a new preclinical model will facilitate molecular investigations on GO and evaluation of new therapeutic interventions.
BACKGROUND: Chronic lung allograft dysfunction (CLAD) is a heterogeneous condition. Characterization of CLAD phenotypes is essential to enhance the understanding of pathogenesis and guide new therapies. The study objective was to validate the new International Society for Heart and Lung Transplantation (ISHLT) CLAD classification system and further explore patients who do not fall into the defined CLAD sub-categories. METHODS: We performed a single-center, retrospective cohort study of adult, first, bilateral lung transplants performed from 2010 to 2015. Patients with CLAD were classified on the basis of the 2019 ISHLT consensus document. CLAD phenotypes and other potential predictors of survival after CLAD onset were assessed using Kaplan−Meier and Cox proportional hazards models. RESULTS: Among the 174 subjects with CLAD, 104 (59.8%) had bronchiolitis obliterans syndrome (BOS), 16 (9.2%) restrictive allograft syndrome (RAS), 9 (5.2%) mixed, and 19 (10.9%) undefined phenotype. A total of 26 patients (14.9%) did not match any of these 4 categories and remained unclassified. Allograft survival post-CLAD onset was longer for patients with BOS (median, 500 days) than patients with RAS (median, 372 days) or mixed (median, 328 days). The 45 patients (26.8%) with undefined/unclassified phenotype were combined and recategorized on the basis of the presence or absence of characteristic RAS-like opacities on chest imaging; those with RAS-like opacities had significantly worse allograft survival than patients with BOS (hazard ratio, 2.14; 95% confidence interval, 1.17−3.93; p = 0.014) and similar survival to RAS or mixed phenotype.
BackgroundVariation in induced models of autoimmunity has been attributed to the housing environment and its effect on the gut microbiota. In Graves’ disease (GD), autoantibodies to the thyrotropin receptor (TSHR) cause autoimmune hyperthyroidism. Many GD patients develop Graves’ orbitopathy or ophthalmopathy (GO) characterized by orbital tissue remodeling including adipogenesis. Murine models of GD/GO would help delineate pathogenetic mechanisms, and although several have been reported, most lack reproducibility. A model comprising immunization of female BALBc mice with a TSHR expression plasmid using in vivo electroporation was reproduced in two independent laboratories. Similar orbital disease was induced in both centers, but differences were apparent (e.g., hyperthyroidism in Center 1 but not Center 2). We hypothesized a role for the gut microbiota influencing the outcome and reproducibility of induced GO.ResultsWe combined metataxonomics (16S rRNA gene sequencing) and traditional microbial culture of the intestinal contents from the GO murine model, to analyze the gut microbiota in the two centers. We observed significant differences in alpha and beta diversity and in the taxonomic profiles, e.g., operational taxonomic units (OTUs) from the genus Lactobacillus were more abundant in Center 2, and Bacteroides and Bifidobacterium counts were more abundant in Center 1 where we also observed a negative correlation between the OTUs of the genus Intestinimonas and TSHR autoantibodies. Traditional microbiology largely confirmed the metataxonomics data and indicated significantly higher yeast counts in Center 1 TSHR-immunized mice. We also compared the gut microbiota between immunization groups within Center 2, comprising the TSHR- or βgal control-immunized mice and naïve untreated mice. We observed a shift of the TSHR-immunized mice bacterial communities described by the beta diversity weighted Unifrac. Furthermore, we observed a significant positive correlation between the presence of Firmicutes and orbital-adipogenesis specifically in TSHR-immunized mice.ConclusionsThe significant differences observed in microbiota composition from BALBc mice undergoing the same immunization protocol in comparable specific-pathogen-free (SPF) units in different centers support a role for the gut microbiota in modulating the induced response. The gut microbiota might also contribute to the heterogeneity of induced response since we report potential disease-associated microbial taxonomies and correlation with ocular disease.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0478-4) contains supplementary material, which is available to authorized users.
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