Graves' Orbitopathy Pathogenesis Research Articles

Role of genetics and epigenetics in Graves' orbitopathy.

Objectives The pathogenesis of Graves' orbitopathy (GO) remains to be fully elucidated. Here we reviewed the role of genetics and epigenetics. Design We conducted a PubMed search with the following key words: Graves' orbitopathy, thyroid eye disease; or Graves' ophthalmopathy; or thyroid-associated ophthalmopathy; and: genetic, or epigenetic, or gene expression, or gene mutation, or gene variant, or gene polymorphism, or DNA methylation, or DNA acetylation. Articles in which whole DNA and/or RNA sequencing, proteome and methylome analysis were performed were chosen. Results The different prevalence of GO in the two sexes as well as racial differences suggest that genetics play a role in GO pathogenesis. In addition, the long-lasting phenotype of GO and of patient-derived orbital fibroblasts suggest a genetic or epigenetic mechanism. Although no genes have been found to confer a specific risk for GO, differential gene expression has been reported in orbital fibroblasts from GO patients vs control fibroblasts, suggesting that an epigenetic mechanism may be involved. In this regard, a different degree of DNA methylation, which affects gene expression, has been found between GO and control fibroblasts, which was confirmed by whole methylome analysis. Histone acetylation and deacetylation, which also affect gene expression, remain to be investigated. Conclusions Although pathogenetic gene variants have not been reported, epigenetic mechanisms elicited by an initial autoimmune insult seem to be needed for differential gene expression to occur and, thus, for GO to develop and persist over time.

Role of Inositol-Requiring Enzyme 1 and Autophagy in the Pro-Fibrotic Mechanism Underlying Graves' Orbitopathy.

Orbital fibroblasts play key roles in the pathogenesis of Graves' orbitopathy (GO), and previous findings have shown that endoplasmic reticulum (ER) stress and autophagy also contribute to GO. In this study, we investigated the presently unclear roles of inositol-requiring enzyme 1 (IRE1) and related autophagy processes in the pro-fibrotic mechanism of GO. Orbital adipose/connective tissues were obtained from eight GO patients and six normal individuals during surgery. GO fibroblasts were transfected with IRE1 small-interfering RNA and treated with bafilomycin A1 (Baf-A1) to evaluate the inhibitory effects of ER stress and autophagy, and protein-expression levels were analyzed through western blotting after stimulation with transforming growth factor (TGF)-β. TGF-β stimulation upregulated IRE1 in GO orbital fibroblasts, whereas silencing IRE1 suppressed fibrosis and autophagy responses. Similarly, Baf-A1, an inhibitor of late-phase autophagy, decreased the expression of pro-fibrotic proteins. IRE1 mediates autophagy and the pro-fibrotic mechanism of GO, which provides a more comprehensive interpretation of GO pathogenesis and suggests potential therapeutic targets.

Changes of plasma cytokines content in Graves’ orbitopathy patients, could be potential markers

Aims: The purpose of this study was to detect potential cytokines markers involved in the Graves’ orbitopathy (GO). Materials and Methods: We measured 25 plasma cytokines using label-free quantitative proteomic analysis in the pilot cohort. In validation cohort, NORCH3, TBP, α-enolase (ENO1), GPX3, ORM1, extracellular matrix protein-1 (ECM1), SERPINE, IGF1, IGKV1, and SPARC were validated using ELISA kits. We used label-free liquid chromatography–mass spectrometry/MS (LC–MS/MS) quantitative proteomic to analyze plasma cytokines content of Graves’ disease and GO patients. Results: The validation cohort showed that the concentrations of ECM1, ENO1, and GPX3, (244.4276 pg/ml, 4.361807 ng/ml, and 17.72794 pmol/ml, respectively) were significantly lower in GO patients samples than those in Graves’ patients (257.3197 pg/ml, 4.601592 ng/ml, and 21.33118 pmol/ml, respectively). Both univariate and multivariate logistic analyses indicated that GPX3, ECM1, and ENO might be predictors of Graves’ ophthalmopathy. A receiver-operating characteristic curve with an area under the curve of 0.938, a sensitivity of 84.4%, and a specificity of 90.5% underscored the applicability of this method. Conclusions: ECM1, ENO1, and GPX3 were associated with the presence of the GO group and may be good biomarkers that play an important role in the pathogenesis of GO.

Unveiling Modern Strategies for Diagnosing and Treating Grave Orbitopathy

Graves' Orbitopathy (GO), also known as thyroid eye disease or thyroid-associated orbitopathy is characterized by inflammation, ocular muscle hypertrophy, adipogenesis, and oedema (due to glycosaminoglycan accumulation). This condition leads to remodeling, tissue expansion, and/or fibrosis within the fibroadipose tissue or extraocular muscles of the orbit. GO manifests as an extrathyroidal aspect of autoimmune thyroid diseases in both Grave's disease (GD) and Hashimoto's thyroiditis.
 The pathophysiological foundation of GO entails the infiltration of B cells, T cells, and CD34+ fibroblasts in the orbit. B cells generate IGF (insulin-like growth factor), wherein IGF and TRab (Thyroid-Receptor antibodies) stimulate the IGF receptor complex and thyrotropin receptor (respectively) on the surface of CD34+ cells which triggers orbital tissue expansion, orbital protrusion, optic nerve compression, and eyeball displacement, resulting in exophthalmos.
 GO progress through an active phase (characterized by inflammation with visible manifestations), followed by a plateau phase (stabilization of GO manifestations), and a gradual resolution of distinctive residual signs and symptoms (inactive phase). This entire process spans between 18-24 months in untreated patients, where disease manifestations significantly depend on the phase during which the disease is identified.
 GO therapy is intended to shorten the active phase and supress its residual eye manifestations during the inactive phase. In general, GO therapy is categorized into general and disease severity-specific approaches.
 GO therapy often falls short of providing satisfactory results, prompting need of surgery to address lingering clinical manifestations. This review presents the latest insights into the pathogenesis and treatment of GO for better management and outcomes.

Determinants of clinical outcome in patients with moderate/severe Graves' orbitopathy undergoing treatment with parenteral glucocorticoids: a retrospective study.

Graves' orbitopathy (GO) occurs in approximately 25-40% of patients with Graves' disease (GD). High levels of anti-thyrotropin receptor antibodies (TRAbs), smoking habit, sex, older age, longer duration and amount of hyperthyroidism or hypothyroidism are well-recognized risk factors for the occurrence, severity and clinical course of GO. Oxidative stress (OX) has recently been shown to play a role in the pathogenesis of GO, and several clinical conditions related to OX have been investigated regarding the presentation and severity of GO. We aimed to evaluate the impact of clinical conditions related to oxidative stress on the outcome of intravenous glucocorticoid (ivGCs) therapy in a cohort of patients with active moderate to severe GO (AMS-GOs) treated at a single institution. We retrospectively studied a series of patients with AMS-GOs who were treated with ivGCs from January 2013 to May 2022. GO clinical evaluation was performed at baseline and at 6 (W6), 12 (W12) and 24 (W24) weeks after starting ivGCs by the seven-point clinical activity score (CAS) alone and by overall clinical criteria (CI) according to the European Group of Graves' Ophthalmopathy (EUGOGO). Total cholesterol and calculated LDL cholesterol (LDLc), triglyceride, body mass index (BMI), diabetes status, history of hypertension (HoH), smoking status, age and sex were used as covariates for the clinical outcome of GO to ivGCs. LDLc and HoH negatively and independently modulated the response of AMS-GOs to ivGCs. Notably, slightly elevated LDLc levels (> 130 mg/dl) reduced the response of orbital soft tissue to ivGCs, whereas more elevated LDLc levels (from 175 mg/dl to 190 mg/dl) and HoH were associated with poorer clinical response of eye motility and proptosis.

Impact of ibrutinib on inflammation in a mouse model of Graves' orbitopathy.

Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO). Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting. In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1β, IL-6, transforming growth factor-β1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group. The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.

Phospholipase C-γ as a Potential Therapeutic Target for Graves' Orbitopathy.

Phospholipase C-γ (PLC-γ) plays a crucial role in immune responses and is related to the pathogenesis of various inflammatory disorders. In this study, we investigated the role of PLC-γ and the therapeutic effect of the PLC-specific inhibitor U73122 using orbital fibroblasts from patients with Graves' orbitopathy (GO). The expression of phospholipase C gamma 1 (PLCG1) and phospholipase C gamma 2 (PLCG2) was evaluated using polymerase chain reaction in GO and normal orbital tissues/fibroblasts. The primary cultures of orbital fibroblasts were treated with non-toxic concentrations of U73122 with or without interleukin (IL)-1β to determine its therapeutic efficacy. The proinflammatory cytokine levels and activation of downstream signaling molecules were determined using Western blotting. PLCG1 and PLCG2 mRNA expression was significantly higher in GO orbital tissues than in controls (P<0.05). PLCG1 and PLCG2 mRNA expression was significantly increased (P<0.05) in IL-1β, tumor necrosis factor-α, and a cluster of differentiation 40 ligand-stimulated GO fibroblasts. U73122 significantly inhibited the IL-1β-induced expression of proinflammatory molecules, including IL-6, IL-8, monocyte chemoattractant protein-1, cyclooxygenase-2, and intercellular adhesion molecule-1 (ICAM-1), and phosphorylated protein kinase B (p-Akt) and p38 (p-p38) kinase in GO fibroblasts, whereas it inhibited IL-6, IL-8, and ICAM-1, and p-Akt and c-Jun N-terminal kinase (p-JNK) in normal fibroblasts (P<0.05). PLC-γ-inhibiting U73122 suppressed the production of proinflammatory cytokines and the phosphorylation of Akt and p38 kinase in GO fibroblasts. This study indicates the implications of PLC-γ in GO pathogenesis and its potential as a therapeutic target for GO.

Yes-Associated Protein Mediates the Transition from Inflammation to Fibrosis in Graves' Orbitopathy.

Background: In Graves' orbitopathy (GO), localized orbital inflammation within the fixed orbit often leads to a fibrotic phenotype resulting in restrictive myopathy or refractory proptosis. However, the molecular pathways related to the transition from inflammation to fibrosis in GO are less understood. Yes-associated protein (YAP) and its homolog, transcriptional coactivator with PDZ-binding motif (TAZ; a Hippo pathway effector), are critical mechanosensors of mechanical stimuli and activate signaling cascades for cell proliferation, differentiation, and transformation. In this study, we aimed to examine the role of YAP in both inflammatory and fibrotic GO pathogenesis. Methods: Based on RNA sequencing performed on freshly obtained orbital adipose tissue from patients with GO and healthy individuals, Gene Ontology analysis and gene set-enrichment analysis were performed to analyze gene-expression differences between GO and normal orbital tissues. The role of YAP in GO-related inflammation and fibrosis was studied in primary cultured orbital fibroblasts. The effects of interleukin-1β (IL-1β)-induced inflammation and transforming growth factor-beta (TGF-β)-induced fibrosis on YAP expression were evaluated using real-time polymerase chain reaction and Western blotting analyses. The effects of YAP on inflammatory and fibrotic responses were also examined by YAP silencing or treatment with pharmacological YAP inhibitors. Results: RNA sequencing revealed enhanced YAP expression in GO orbital tissues. Gene Ontology analysis indicated that "response to mechanical stimulus"-related genes were overexpressed in GO orbital tissues, along with those enriched for the "adipose proliferation," "inflammatory responses," and "hormone stimulus responses" terms. IL-1β did not enhance YAP expression, and YAP silencing decreased IL-1β-induced IL-6 expression while increasing prostaglandin-endoperoxide synthase 2 expression, leading to paradoxical pro-inflammatory effects. Conversely, TGF-β enhanced YAP expression, and YAP silencing and pharmacological YAP inhibitor (cerivastatin, verteporfin, TED-347, and CA3) treatment significantly reduced TGF-β-induced myofibroblast differentiation and collagen formation. Conclusion: YAP, a mechanotransducer responding to mechanical stimuli, was strongly expressed in GO orbital tissues, and YAP was induced by TGF-β in orbital fibroblasts. Our study establishes YAP as a novel mediator of GO pathobiology, potentially mediating the transition from early inflammation to chronic fibrosis in GO. The finding that YAP inhibition suppressed TGF-β-induced fibrotic response suggests YAP as a therapeutic target against the fibrotic mechanism of GO.

The Role of Adipsin, Complement Factor D, in the Pathogenesis of Graves' Orbitopathy.

Graves' orbitopathy (GO) is an orbital manifestation of autoimmune Graves' disease, and orbital fibroblast is considered a target cell, producing pro-inflammatory cytokines and/or differentiating into adipocytes. Adipose tissue has been focused on as an endocrine and inflammatory organ secreting adipokines. We investigated the pathogenic role of a specific adipokine, adipsin, known as complement factor D in Graves' orbital fibroblasts. The messenger RNA (mRNA) expression of multiple adipokines was investigated in adipose tissues harvested from GO and healthy subjects. Adipsin protein production was analyzed in primary cultured orbital fibroblasts under insulin growth factor (IGF)-1, CD40 ligand (CD40L) stimulation, and adipogenesis. The effect of blocking adipsin with small interfering RNA (siRNA) on pro-inflammatory cytokine production and adipogenesis was evaluated using quantitative real-time PCR, Western blot, and ELISA. Adipogenic differentiation was identified using Oil Red O staining. Adipsin gene expression was significantly elevated in GO tissue and increased after the stimulation of IGF-1 and CD40L, as well as adipocyte differentiation in GO cells. Silencing of adipsin suppressed IGF-1-induced IL-6, IL-8, COX2, ICAM-1, CCL2 gene expression, and IL-6 protein secretion. Adipsin suppression also attenuated adipocyte differentiation. Exogenous treatment of recombinant adipsin resulted in the activation of the Akt, ERK, p-38, and JNK signaling pathways. Adipsin, secreted by orbital fibroblasts, may play a distinct role in the pathogenesis of GO. Inhibition of adipsin ameliorated the production of pro-inflammatory cytokines and adipogenesis in orbital fibroblasts. Our study provides an in vitro basis suggesting adipsin as a potential therapeutic target for GO treatment.

A review of TSHR- and IGF-1R-related pathogenesis and treatment of Graves' orbitopathy.

Graves' orbitopathy (GO) is an organ-specific autoimmune disease, but its pathogenesis remains unclear. There are few review articles on GO research from the perspective of target cells and target antigens. A systematic search of PubMed was performed, focusing mainly on studies published after 2015 that involve the role of target cells, orbital fibroblasts (OFs) and orbital adipocytes (OAs), target antigens, thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R), and their corresponding antibodies, TSHR antibodies (TRAbs) and IGF-1R antibodies (IGF-1R Abs), in GO pathogenesis and the potentially effective therapies that target TSHR and IGF-1R. Based on the results, OFs may be derived from bone marrow-derived CD34+ fibrocytes. In addition to CD34+ OFs, CD34- OFs are important in the pathogenesis of GO and may be involved in hyaluronan formation. CD34- OFs expressing Slit2 suppress the phenotype of CD34+ OFs. β-arrestin 1 can be involved in TSHR/IGF-1R crosstalk as a scaffold. Research on TRAbs has gradually shifted to TSAbs, TBAbs and the titre of TRAbs. However, the existence and role of IGF-1R Abs are still unknown and deserve further study. Basic and clinical trials of TSHR-inhibiting therapies are increasing, and TSHR is an expected therapeutic target. Teprotumumab has become the latest second-line treatment for GO. This review aims to effectively describe the pathogenesis of GO from the perspective of target cells and target antigens and provide ideas for its fundamental treatment.

Current concepts regarding Graves' orbitopathy.

Graves' orbitopathy (GO) is an orbital autoimmune disorder and the main extrathyroidal manifestation of Graves' disease, the most common cause of hyperthyroidism. GO affects about 30% of Graves' patients, although fewer than 10% have severe forms requiring immunosuppressive treatments. Management of GO requires a multidisciplinary approach. Medical therapies for active moderate-to-severe forms of GO (traditionally, high-dose glucocorticoids) often provide unsatisfactory results, and subsequently surgeries are often needed to cure residual manifestations. The aim of this review is to provide an updated overview of current concepts regarding the epidemiology, pathogenesis, assessment, and treatment of GO, and to present emerging targeted therapies and therapeutic perspectives. Original articles, clinical trials, systematic reviews, and meta-analyses from 1980 to 2021 were searched using the following terms: Graves' disease, Graves' orbitopathy, thyroid eye disease, glucocorticoids, orbital radiotherapy, rituximab, cyclosporine, azathioprine, teprotumumab, TSH-receptor antibody, smoking, hyperthyroidism, hypothyroidism, thyroidectomy, radioactive iodine, and antithyroid drugs. Recent studies suggest a secular trend toward a milder phenotype of GO. Standardized assessment at a thyroid eye clinic allows for a better general management plan. Treatment of active moderate-to-severe forms of GO still relies in most cases on high-dose systemic-mainly intravenous-glucocorticoids as monotherapy or in combination with other therapies-such as mycophenolate, cyclosporine, azathioprine, or orbital radiotherapy-but novel biological agents-including teprotumumab, rituximab, and tocilizumab-have achieved encouraging results.

Orbital Signaling in Graves' Orbitopathy.

Graves’ orbitopathy (GO) is a complex and poorly understood disease in which extensive remodeling of orbital tissue is dominated by adipogenesis and hyaluronan production. The resulting proptosis is disfiguring and underpins the majority of GO signs and symptoms. While there is strong evidence for the thyrotropin receptor (TSHR) being a thyroid/orbit shared autoantigen, the insulin-like growth factor 1 receptor (IGF1R) is also likely to play a key role in the disease. The pathogenesis of GO has been investigated extensively in the last decade with further understanding of some aspects of the disease. This is mainly derived by using in vitro and ex vivo analysis of the orbital tissues. Here, we have summarized the features of GO pathogenesis involving target autoantigens and their signaling pathways.

Role of the mononuclear cell infiltrate in Graves' orbitopathy (GO): results of a large cohort study.

The extent to which mononuclear cells and TSH-receptor autoantibodies (TRAb) contribute to Graves' orbitopathy (GO) is not completely defined. Here we investigated the relationship between the immunohistochemical phenotype of orbital infiltrating cells and GO features in a large number of patients. We conducted an observational cohort study in 76 consecutive patients with GO (16 men and 60 women) who underwent orbital decompression over a period of 18 consecutive months. An ophthalmological evaluation was performed in all patients, as well as immunohistochemistry for CD3, CD4, CD8, CD56 (T-cell markers), CD25 (T and B-cell marker), CD20, CD19 (B-cell markers), and CD138 (plasmacell marker) in specimens collected at decompressive surgery. Having established cutoff values for each marker, cell infiltrates were found in 60 patients (78.9%; CD3: 39.4%, CD4 55.2%, CD8 50%, CD56: 0%, CD25: 28.9%, CD20: 51.3%, CD19: 25%, CD138: 26.3%). Eleven (14.4%) stained exclusively for CD138 (plasmacells). Patients with CD4-positive mononuclear cells had a significantly greater GO clinical activity score (CAS) (mean difference 1.07, 95% CI - 0.33 to - 1.82, P = 0.004 by univariate, P = 0.05 by multivariate analysis). CAS as well as the remaining GO features were not affected significantly by the mononuclear cell subpopulations in multivariate analyses. Mononuclear cell infiltrates are present in the majority of GO patients, with a small percentage represented exclusively by plasmacells. CD4 cells exert a major role on GO activity. These findings may represent a further advancement in the comprehension of GO pathogenesis.

Peripheral blood mononuclear cells - Can they provide a clue to the pathogenesis of Graves' Orbitopathy?

Graves' disease (GD) is an autoimmune disorder affecting primarily the thyroid gland. The most common extrathyroidal manifestation of GD is known as Graves' orbitopathy (GO). Bone marrow-derived fibrocytes represent a subset of monocytes in peripheral blood mononuclear cells (PBMCs), infiltrate the orbital tissues, and contribute to the pathogenesis of GO. Hence objectives of the study included whether the concentration of fibrocytes in peripheral blood was higher in GO, whether TSHR m RNA expression and TSHR surface expression in peripheral blood were higher in GO in comparison to Graves' Disease (GD) and Control subjects. The percentage of circulating fibrocytes (FC) along with TSHR on its cell surface (CD 34+, CD 45+, CXCR4+, Collagen 1+, TSHR+) were assessed by flow cytometry of 50 patients with GD and GO and 15 healthy donors (Control). TSHR mRNA expression was measured by q RT PCR. The concentration of circulating fibrocytes was significantly higher in GO compared to GD and control [GO 17% vs GD 3% vs control 0.7% (p < 0.05)]. Moreover, these fibrocytes express a significantly higher level of TSHR in GO. This was corroborated by the measure of TSH mRNA; in GD it was 2.3-fold higher and in GO it was 3.9 fold higher than in control, in GO this transcript level was 1.7fold higher than GD (p < 0.05). TSHR+ fibrocytes were significantly positively correlated with CAS (p = 0.004) and negatively correlated with age (p = 0.01) and duration of disease (p = 0.01) in GO. This study sheds further light on the pathogenesis of GO.

Signal transducer and activator of transcription 3 as a potential therapeutic target for Graves’ orbitopathy

The roles of signal transducer and activator of transcription 3 (STAT3) in inflammation, oxidative stress, and adipogenesis during Graves’ orbitopathy (GO) are incompletely understood. Here, STAT3 expression in orbital tissues (from individuals with GO and healthy control subjects) was studied, and the role of STAT3 in GO pathogenesis was examined through small-interfering RNA (siRNA)-mediated silencing in primary orbital fibroblasts. STAT3 mRNA expression was higher in GO orbital tissues than in non-GO tissues. Treatment with proinflammatory cytokines, thyroid-stimulating hormone, or insulin-like growth factor-1 induced STAT3 mRNA in GO orbital fibroblasts, but not in non-GO cells. STAT3 silencing inhibited interleukin-1β-induced inflammatory cytokines and oxidative stress-induced haem oxygenase-1 expression. STAT3 siRNA-transfected GO orbital fibroblasts showed decreased adipocyte differentiation. STAT3 affected proinflammatory cytokine production, oxidative stress responses, and adipogenesis in an in vitro model of GO, suggesting that STAT3 mediates GO pathology, and that modulating STAT3 expression may have therapeutic potential against GO.

Mechanisms That Underly T Cell Immunity in Graves' Orbitopathy.

Graves’ orbitopathy (GO), also known as thyroid-associated ophthalmopathy, is the most common ocular abnormality of Graves’ disease. It is a disfiguring, invalidating, and potentially blinding orbital disease mediated by an interlocking and complicated immune network. Self-reactive T cells directly against thyroid-stimulating hormone receptor-bearing orbital fibroblasts contribute to autoimmune inflammation and tissue remodeling in GO orbital connective tissues. To date, T helper (Th) 1 (cytotoxic leaning) and Th2 (antibody leaning) cell subsets and an emerging role of Th17 (fibrotic leaning) cells have been implicated in GO pathogenesis. The potential feedback loops between orbital native residential CD34- fibroblasts, CD34+ infiltrating fibrocytes, and effector T cells may affect the T cell subset bias and the skewed pattern of cytokine production in the orbit, thereby determining the outcomes of GO autoimmune reactions. Characterization of the T cell subsets that drive GO and the cytokines they express may significantly advance our understanding of orbital autoimmunity and the development of promising therapeutic strategies against pathological T cells.

PERK mediates oxidative stress and adipogenesis in Graves' orbitopathy pathogenesis.

We examined endoplasmic reticulum (ER) stress-related gene expression in orbital tissues from patients with Graves' orbitopathy (GO) and the effects of silencing protein kinase RNA-like endoplasmic reticulum kinase (PERK) in primary orbital fibroblast cultures to demonstrate the therapeutic potential of PERK-modulating agents in GO management. The expression of ER stress-related genes in orbital tissue harvested from individuals with or without GO was studied using real-time PCR. The role of PERK in GO pathogenesis was examined through small-interfering RNA (siRNA)-mediated silencing in cultured primary orbital fibroblasts. Intracellular reactive oxygen species (ROS) levels induced in response to cigarette smoke extract (CSE) or hydrogen peroxide were measured using 5-(and 6)-carboxy-20,70-dichlorodihydrofluorescein diacetate staining and flow cytometry. Cells were stained with Oil Red O, and adipogenesis-related transcription factor expression was evaluated through Wwestern blotting after adipogenic differentiation. PERK, activating transcription factor 4 (ATF4), and CCAAT-enhancer-binding protein (C/EBP)-homologous protein (CHOP) mRNA levels were significantly higher in GO orbital tissues than in non-GO orbital tissues. PERK silencing inhibited CSE- or hydrogen peroxide-induced ROS generation. After adipogenic differentiation, GO orbital fibroblasts revealed decreased lipid droplets and downregulation of C/EBPα, C/EBPβ, and peroxisome proliferator-activator gamma (PPARγ) in PERK siRNA-transfected cells. The orbital tissues of patients with GO were exposed to chronic ER stress and subsequently exhibited enhanced unfolded protein response (especially through the PERK pathway). PERK silencing reduced oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. Our results imply that PERK-modulating agents can potentially be used to manage GO.

Autoantibodies, immunoglobulins and cytokine profile in patients with graves' disease and Graves' orbitopathy

Graves' Orbitopathy (GO) - also known as Thyroid Eye Disease (TED) - is an autoimmune condition in the modern sense. It is closely associated with autoimmune thyroid diseases. Cytokine-mediated mechanisms play a critical part in immunopathogenesis of autoimmune thyroid diseases including GO. Investigating cytokine profiles as well as antibodies to tissue-specific antigens is essential for explaining GO pathogenesis and developing future therapeutic strategies. The study examines serum levels of cytokines, autoantibodies and immunoglobulins IgG and IgG4 as mediators of autoimmune inflammation in patients with GO and Graves' Disease (GD). The study included 52 patients (104 orbits) aged 25-70 years (mean age 48,8±12,3) in the active phase of GO and GD verified with the international diagnostic standards. These patients did not get any treatment for GO before. The control group consisted of 14 individuals (28 orbits) aged 30-68 years without known autoimmune disease.Serum levels of IgG, IgG4,TNFα, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-13, sIL-6R, sTNFα- RI и TNFα- R2 IL-2R, TGFβ1, TGF β3, antibodies to TSH-receptor, free T4, free T3 and TSH were measured. A diagnostic ultrasound exam of thyroid gland, multislice computed tomography (MSCT) / magnetic resonance imaging (MRI) of orbits were performed. Mean duration of GO prior to being admitted to the centre was 8,8±1,5 months (range: 1 - 48 months). According to the degree of thyrotoxicosis compensation: 24 patients were clinically euthyroid, TSH 3,3±0,7 mU/L, free T4 11,9±0,59 pmol/L, free T3 3,97±0,1 pmol/L; 28 patients were considered to have subclinical thyrotoxicosis: TSH 0,03±0,01 mU/L, free T4 14,2±1,0 pmol/L, free T3 5,77±0,49 pmol/L. Serum levels of sTNFα-R2 (p=0,041, p≤0,05), sIL-2R (p=0,020, p≤0,05), TGFβ1 (p=0,000, p≤0,001) were significantly higher in patients with GO compared to the control group. Serum levels of sTNFRα2 (p=0,038, p&lt;0,05) and TGFβ1 (P=0,011, p≤0,05) were positively correlated with the duration of GO. The positive correlations between the serum level of sIL-6R (p=0,034, p≤0,05) and the severity of GO as well as between the serum level of sTNFα- R 1 (P=0,012, p≤0,05) and activity of GO were observed. 54% of patients had elevated concentration level of IgG4 in IgG ( &gt;5%). High levels of soluble cytokine receptors sTNFα-R2 and sIL-2R and cytokine TGFβ1 in patients with long-standing untreated GO and GD being euthyroid or having subclinical thyrotoxicosis indicate activation of regulatory T cells aimed at suppressing autoimmune processes. High concentration level of IgG4 in IgG and cytokine TGFβ1 can determine the development of fibrotic changes in the orbital tissues. A decrease in the concentration of cytokine TGFβ1 can indicate an unfavorable course of the disease GO.

Antioxidant Therapy in Graves' Orbitopathy.

The balance of the cell redox state is a key point for the maintenance of cellular homeostasis. Increased reactive oxygen species (ROS) generation leads to oxidative damage of tissues, which is involved in the development of several diseases, including autoimmune diseases. Graves’ Orbitopathy (GO) is a disfiguring autoimmune-related condition associated with Graves’ Disease (GD). Patients with active, moderate-to-severe GO, are generally treated with high doses intravenous glucocorticoids (ivGCs) and/or orbital radiotherapy. On the contrary, up to recently, local ointments were the treatment most frequently offered to patients with mild GO, because the risks related to ivGCs does not justify the relatively poor benefits expected in mild GO. However, a medical treatment for these patients is heavily wanted, considering that GO can progress into more severe forms and also patients with mild GO complain with an impairment in their quality of life. Thus, based on the role of oxidative stress in the pathogenesis of GO, a therapy with antioxidant agents has been proposed and a number of studies have been performed, both in vitro and in vivo, which is reviewed here.

Role of binding immunoglobulin protein (BiP) in Graves' orbitopathy pathogenesis.

Inflammation and adipogenesis represent the main pathogenic mechanisms of Graves' orbitopathy (GO), and oxidative stress is a well-known inducer of GO pathology. Endoplasmic reticulum (ER) stress has been suggested as a major contributor to inflammation and reactive oxygen species (ROS) generation. In this study, we investigated the role of the ER-stress chaperone protein, binding immunoglobulin protein (BiP), in GO pathogenesis. Using primary cultures of orbital fibroblasts from patients with GO, we examined the role of BiP in GO pathogenesis by silencing its expression with small-interfering RNA (siRNA). Inflammatory cytokine expression was analysed by Western blotting and ELISA. Intracellular ROS levels induced by hydrogen peroxide or cigarette smoke extract were measured by 5-(and 6)-carboxy-20,70-dichlorodihydrofluorescein diacetate staining and flow cytometry. After adipogenic differentiation in BiP siRNA-transfected cells, the cells were stained with Oil Red O, and the levels of adipogenic transcription factors were determined by Western blot analysis. BiP mRNA expression levels were significantly higher in GO orbital tissues than in non-GO orbital tissues. Silencing BiP attenuated the expression of pro-inflammatory cytokines (interleukin-6, intercellular adhesion molecule-1, and monocyte chemotactic protein-1) in primary cultured GO orbital fibroblasts. Silencing BiP also reduced ROS generation, hyaluronan production, and adipocyte differentiation. These findings suggest that ER stress is involved in the aetiology of GO and that modulation of ER stress has therapeutic potential for GO.