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.

SENSITIVITY OF THREE THYROTROPIN RECEPTOR ANTIBODY ASSAYS IN THYROID-ASSOCIATED ORBITOPATHY

Background: Thyrotropin receptor autoantibodies (TSH-R-Ab) are indispensable biomarkers in the laboratory assessment of thyroid-associated orbitopathy (TAO). Clinical sensitivity of three different assays for TSH-R-Ab determination was evaluated in patients with TAO. Methods: 87 consecutive TAO patients were enrolled and their serum samples analyzed in parallel with three assays. An ECLIA competitive binding and a chemiluminescent bridge immunoassay were used to measure total and binding TSH-R-Ab concentration, while their functional activity was determined using a stimulatory TSH-R-Ab (TSAb) cell-based bioassay. Results: Compared to the two binding assays (ECLIA p<0.001, bridge p=0.003), the TSAb bioassay was more sensitive pertaining to the positive detection of TSH-R-Ab in TAO patients. No difference (p=0.057) was noted between the ECLIA and bridge assays regarding sensitivity rate. All patients with active and/or moderate-to-severe TAO tested positive in the TSAb bioassay (100% and 100%, respectively), while the positivity rates for bridge and ECLIA binding assays were 89.7% and 82.1% for active TAO, and 90.2% and 86.3% for severe TAO, respectively. Negative predictive values of the bioassay, bridge, and ECLIA assays were 100%, 75%, and 71%, respectively for active TAO, and 100%, 86%, and 71%, respectively for moderate-to-severe TAO. The superiority of the bioassay was most prominent in euthyroid (ET) TAO. Positivity rates of the TSAb bioassay, bridge and ECLIA binding assays were 89.6%, 75%, and 64.6%, respectively for inactive TAO; 86.1%, 69.4%, and 52.8%, respectively for mild TAO; 87.5%, 62.5%, and 12.5%, respectively for euthyroid TAO. The bridge assay correlated better with the ECLIA binding assay (ρ=0.893, p<0.001), compared to the bioassay (ρ=0.669, p<0.001). Conclusions: In patients with TAO of various activity and severity, the TSAb bioassay demonstrates a superior clinical performance compared to both ECLIA and bridge binding assays.

The effect of radioiodine treatment on the characteristics of TRAb in Graves’ disease

Background Graves’ disease (GD) is one of the most common autoimmune thyroid diseases (AITDs) in humans, and thyrotropin receptor antibody (TRAb) is a characterized autoantibody in GD. The use of radioactive iodine therapy (RAI) for GD treatment is increasing. Objectives We studied the biological properties of TRAb and evaluated the effect of RAI therapy on TRAb in GD patients. Methods In total, 225 patients (22 onset GD patients without 131I therapy, 203 GD patients treated with 131I therapy) and 20 healthy individuals as normal controls were included in this study. Clinical assessments were performed, and we examined in vitro the biological properties of TRAb in the 22 onset GD patients and 20 controls as well as 84 GD patients with 131I therapy. Results Serum TRAb and thyroid peroxidase antibody (TPOAb) levels increased in the initial year of RAI treatment, and both antibodies decreased gradually after one year. After 5 years from radioiodine treatment, TRAb and TPOAb levels decreased in 88% and 65% of GD patients, respectively. The proportion of patients positive for thyroid-stimulatory antibody (TSAb) was significantly higher in the 7–12-month group, and thyroid-blocking antibody (TBAb) levels were elevated after one year in half of the patients who received 131I treatment. Conclusions Treatment of GD patients with radioiodine increased TPOAb and TRAb (their main biological properties were TSAbs) within the first year after therapy, and the main biological properties of elevated TRAb were TBAbs after 1 year.

Probiotic Bifidobacterium longum supplied with methimazole improved the thyroid function of Graves’ disease patients through the gut-thyroid axis

Graves’ disease (GD) is an autoimmune disorder that frequently results in hyperthyroidism and other symptoms. Here, we designed a 6-month study with patients divided into three treatment groups, namely, methimazole (MI, n = 8), MI + black bean (n = 9) and MI + probiotic Bifidobacterium longum (n = 9), to evaluate the curative effects of probiotics supplied with MI on thyroid function of patients with GD through clinical index determination and intestinal microbiota metagenomic sequencing. Unsurprisingly, MI intake significantly improved several thyroid indexes but not the most important thyrotropin receptor antibody (TRAb), which is an indicator of the GD recurrence rate. Furthermore, we observed a dramatic response of indigenous microbiota to MI intake, which was reflected in the ecological and evolutionary scale of the intestinal microbiota. In contrast, we did not observe any significant changes in the microbiome in the MI + black bean group. Similarly, the clinical thyroid indexes of patients with GD in the probiotic supplied with MI treatment group continued to improve. Dramatically, the concentration of TRAb recovered to the healthy level. Further mechanistic exploration implied that the consumed probiotic regulated the intestinal microbiota and metabolites. These metabolites impacted neurotransmitter and blood trace elements through the gut-brain axis and gut-thyroid axis, which finally improved the host’s thyroid function.

Evaluation of choroidal circulation and stromal features in Graves’ disease

Purpose: This study aimed to evaluate choroidal thickness (CT) and choroidal vascularity index (CVI) in patients with Graves’ disease (GD) without ocular involvement. Methods: Fifty patients diagnosed with GD and 50 age and gender matched healthy control subjects were retrospectively evaluated. Measurements were taken from five different points on CT images. Choroid images were classified as lumen regions (LA) and stromal regions (SA) using the image binarization method. CVI was calculated by dividing LA by the total choroidal area (TCA). The effects of Thyrotropin Receptor Antibody (TRAb), age, GD duration, blood pressure, axial length measurements, and intraocular pressure were analyzed on CT and CVI measurements. Results: Mean age was 40.1 ± 13.5 years in the patient group and 39.3 ± 13.6 years in the control group ( p = 0.89). There was no significant difference between the GD group and control group in terms of CT measurements. There was a significant difference between the mean CVI measurements of the GD group and control group (68.03 ± 3.41 and 66.62 ± 3.11, respectively) ( p < 0.001). Univariate linear regression analysis revealed a positive correlation between TRAb and CVI ( p = 0.013). Conclusion: While there was no significant difference between the CT measurements of the GD group and the control group, the GD group had significantly higher CVI measurements.

Expansion of inflammatory monocytes in periphery and infiltrated into thyroid tissue in Graves’ disease

Monocytes are important mediators of immune system and are reported to be altered in autoimmune disorders. Little is known about the pathological role of monocytes in Graves’ disease (GD). Thus, we investigated monocytes in periphery and thyroid tissue in GD. Untreated GD patients were enrolled and followed up until remission. Monocytes were significantly increased and positively correlated with anti-thyrotropin receptor antibody (TRAb) in untreated GD (rcounts = 0.269, P < 0.001; rpercentage = 0.338, P < 0.001). Flow cytometry showed CD14++ CD16+ monocytes were increased and CD14++ CD16- monocytes were decreased in untreated GD (both P < 0.001). Skewed monocyte subsets were recovered in GD with remission. Serum B cell-activating factor (BAFF) was positively correlated with TRAb (r = 0.384 and P = 0.001). CD14++ CD16+ monocytes expressed higher level of BAFF in untreated GD (P < 0.05). The frequency of CD14+ monocytes and CD14+ CD16+ monocytes were significantly higher in GD thyroid tissue than in normal thyroid tissue (both P < 0.001). Our study suggested CD14++ CD16+ monocytes were significantly expanded and involved in the production of TRAb via secreting a higher level of BAFF in periphery. Besides, monocytes infiltrated into thyroid tissue and thus could serve as an important participant in GD pathogenesis.

Correlation between Interleukin-4 Gene Promoter Polymorphisms with Thyrotropin Receptor Antibody and Transforming Growth Factor-β

AIM: The aim of this study was to determine the correlation between interleukin-4 (IL-4) gene promoter polymorphisms with thyrotropin receptor antibody (TRAb) and transforming growth factor-β (TGF-β). METHODS: This study was conducted from August 2015 until December 2015 in the internal medicine department in Dr. M. Djamil Hospital, Padang, West Sumatera, Indonesia. Graves’ disease was confirmed by measuring free thyroxine, thyroid-stimulating hormone, and TRAb. We examined that IL-4 promotor gene polymorphism was examined with a polymerase chain reaction. Graves’ disease serum patients will be used to check levels of TGFβ and TRAb antibodies using the enzyme-linked immunoassay method. RESULTS: There are 15 patients in this study. The average of age in patients group is 40.87 (11.23) years. The number of female patients in this study is more than male patients, with the percentage of women are 73.3%, and men are 26.7%. The sequencing examination on IL-4 gene promoter resulted in 2 single nucleotide polymorphism motifs, which are rs2243250 and rs2070847. The mean TRAb level in wild type and mutant group is 6.77 (5.73) IU/L and 4.66 (3.91) IU/L, respectively. The mean TGF-β levels in wild type and mutant group are 1168.89 (438.91) pg/mL and 1114.79 (296.02) pg/mL, respectively. Statistical tests showed no association between IL-4 gene promoter polymorphisms with TRAb and TGF-β levels (p > 0.05). CONCLUSION: There is no correlation between IL-4 gene promoter polymorphisms with TRAb and TGF-β.