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.

Comparative assessment of gut microbial composition and function in patients with Graves' disease and Graves' orbitopathy.

BACKGROUND: A previous study indicated that gut microbiota changed notably in Graves' orbitopathy (GO) patients as compared to controls. However, the characteristics of intestinal bacteria in Graves' disease (GD) and GO are unclear. OBJECTIVE: The present study aimed to identify specific intestinal bacteria of GD and GO, respectively. METHODS: The gut microbial communities of the fecal samples of 30 GD patients without GO, 33 GO subjects, and 32 healthy subjects were analyzed and compared by 16S rRNA gene sequencing. RESULTS: At the phylum level, the proportion of Deinococcus-Thermus and Chloroflexi was decreased significantly in GO patients as compared to GD. At the genus level, the proportion of Subdoligranulum and Bilophila was increased while that of Blautia, Anaerostipes, Dorea, Butyricicoccus, Romboutsia, Fusicatenibacter, unidentified_ Lachnospiraceae, unidentified_Clostridiales, Collineslla, Intestinibacter, and Phascolarctobacterium was decreased in the GO group as compared to the GD group. Random forest analysis was used for the identification of specific intestinal microbiota, and Deinococcus-Thermus, Cyanobacteria and Chloroflexi were ranked in the top ten according to their contributions to sample classification. Moreover, compared to the control, there were multiple gut bacterial enrichment metabolic pathways in GO and GD patients, including nucleotide metabolism, enzyme family, and energy metabolism. Compared to GO, the only enrichment metabolic pathway found in GD was the viral protein family. CONCLUSIONS: This study highlighted the significant differences in the intestinal microbiota and predictive functions of GD with GO, thereby providing new insights into the role of the gut bacteria that might contribute to the development of GO in GD patients.

Emerging Insights Into the Role of Epigenetics and Gut Microbiome in the Pathogenesis of Graves' Ophthalmopathy.

Graves' Ophthalmopathy (GO) is an organ-specific autoimmune disease that is often characterized by infiltration of orbital tissues and is considered as the most common extra-thyroid manifestation of Graves' disease (GD). Although genetic susceptibility has been found to be critical for the phenotype of GO, the associated risk alleles in a single gene are generally insufficient to cause the disease. Accruing evidence has shown that epigenetic disorders can act as the potentially missing link between genetic risk and clinically significant disease development. Abnormal epigenetic modifications can lead to pro-inflammatory cascades and activation of orbital fibroblasts (OFs) by promoting the various inflammatory response pathways and regulating the diverse signaling molecules that are involved in the fibrogenesis and adipogenesis, thereby leading to the significant expansion of orbital tissues, fibrosis and inflammation infiltration. Additionally, emerging evidence has shown that the gut microbiome can possibly drive the pathogenesis of GO by influencing the secretion of Thyrotropin receptor antibody (TRAb) and T-helper 17 (Th17)/regulatory T cells (Treg) imbalance. This paper describes the latest epigenetic research evidence and progress made in comprehending the mechanisms of GO development, such as DNA methylation, histone modification, non-coding RNAs, and the gut microbiome.

Microbial Keratitis in Thyroid Eye Disease: Clinical Features, Microbiological Profile, and Treatment Outcome.

PURPOSE: To report the incidence, clinical features, microbiologic profile, and risk indicators in the development of microbial keratitis in Thyroid Eye Disease (TED). METHODS: All patients who were diagnosed to have TED and developed microbial keratitis between the years 2009 to 2017 at the Ophthalmic Plastic Surgery service, LV Prasad Eye Institute were included in this retrospective interventional study. The clinical features, microbiological profile, and treatment outcome of the infection were studied. Possible risk factors leading to the development of microbial keratitis were studied. RESULTS: A total of 1,000 patients of TED were evaluated in the 10-year period. Of the 1,000, 13 patients (14 eyes, 1.4%) were diagnosed with microbial keratitis. The average age at presentation was 44 years (range 1969 years). Of the 13 patients, 10 (77%) were men, 12 (92%) were hyperthyroid, and 12 (92%) were active (average clinical activity score 3) at presentation. Average exophthalmometry value in the involved eye was 24.75mm, and severe eyelid retraction (>2mm scleral show) was noted in 13 of 14 eyes. None of the patients had optic nerve compression. Moderate motility restriction (2 in all gazes) was noted in 6 eyes, and severe motility restriction (4 in all gazes) in 8 eyes. At presentation, 11 (85%) had visual acuity of counting fingers at 1 meter or less, The mean follow up from the time of presentation was 18.3 months (range 566 months). Majority of the eyes (8/14) presented with severe infection (panophthalmitis with microbial keratits = 1, total corneal infiltrate with/without melt = 4, severe thinning/perforation = 4). Microbiological work up of 14 eyes revealed presence of gram-negative bacteria in 5 eyes which included Escherichia coli, Pseudomonas sp., and Acinetobacter sp., and gram-positive bacteria in 4 eyes including Streptococcus pneumoniae, Corynebacterium sp., and Staphylococcus sp. Three eyes revealed a mixed infection of E. coli with Alternaria sp, and E. coli with Corynebacterioum amycolatum while 1 had Corynebacterium pseudodiptheriticum, and S. pneumoniae. Two eyes of the bilateral case showed no growth. Antibiotic susceptibility revealed 6 of the 7 gram-negative isolates were multidrug resistant, whereas the gram-positive isolates were susceptible to most drugs tested. Surgical procedures required were tarsorrhaphy in 7 eyes, tissue adhesive with bandage contact lens in 4, evisceration in 4, levator recession in 2, 3-wall orbital decompression in 2, and penetrating keratoplasty in 1 eye. The visual acuity at presentation was counting fingers or worse in 10/14 eyes. Posttreatment, 10 eyes achieved resolution of infiltrate (with visual improvement in 2), and 4 required evisceration. CONCLUSIONS: In the authors large series of TED, microbial keratitis was noted in 1.3% of patients presenting to a tertiary eye center. Majority presented with advanced diseases and ended with a poor outcome. Gram-negative isolates showed multidrug resistance. An association with early phase of active TED (CAS 3 or more), severe eyelid retraction, and moderate-severe motility restriction is suggested.Microbial Keratitis occurs in 1.3% cases of Thyroid Eye Disease. It is more common in men, and in active disease. The microbiological spectrum and possible clinical risk factors are presented.

Alterations in the intestinal microbiota of patients with severe and active Graves' orbitopathy: a cross-sectional study.

BACKGROUND: The intestinal microbiota was linked to autoimmune diseases. Graves' orbitopathy (GO) is an autoimmune disease that is usually associated with Graves' disease. However, information on the microbiome of GO patients is yet lacking. OBJECTIVES: To investigate whether GO patients differ from healthy controls in the fecal microbiota. DESIGN: A cross-sectional study. SETTING: 33 patients with severe and active GO and 32 healthy controls of Han nationality were enrolled between March 2017 and March 2018. METHODS: The Gut microbial communities of the fecal samples of GO patients and healthy controls were analyzed and compared by 16S rRNA gene sequencing. RESULTS: Community diversity (Simpson and Shannon) was significantly reduced in fecal samples from patients with GO as compared to controls (p < 0.05). The similarity observed while assessing the community diversity (PCoA) proposed that the microbiota of patients with GO differ significantly from those of controls (p < 0.05). At the phyla levels, the proportion of Bacteroidetes increased significantly in patients with GO (p < 0.05), while at the genus and species levels, significant differences were observed in the bacterial profiles between the two groups (p < 0.05). LIMITATIONS: Single-centered study design and limited fecal samples. CONCLUSIONS: The present study indicated distinctive features of the gut microbiota in GO patients. The study provided evidence for further exploration in the field of intestinal microbiota with respect to the diagnosis and treatment of GO patients by modifying the microbiota profile.