Association between high mobility group box-1 circulation level and Graves' ophthalmopathy

Introduction

Graves' disease is the most common form of hyperthyroidism, occurring in 1% to 1.5% of the general population.¹ The underlying causes and mechanisms of Graves' disease remain elusive.² Autoimmune thyroid disorders like Graves' disease involve complex immune processes.³ In Graves' disease, the autoimmune response affects the orbital fibroblast tissue and triggers inflammation, leading to orbital fibroblast remodeling.⁴ Graves' ophthalmopathy is a hallmark of Graves' disease and an additional thyroid manifestation that is of great importance.⁵

Graves' ophthalmopathy occurs in 23% of cases before diagnosis, 39% after diagnosis, and 37% concurrent with diagnosis.⁶ The exact pathophysiology of Graves' ophthalmopathy is not well understood, with numerous factors influencing its progression and contributing elements.⁷ The involvement of damage-associated molecular patterns (DAMPs), endogenous compounds released during cellular stress and injury, as well as non-apoptotic cell death, may contribute to the inflammation in Graves' ophthalmopathy.⁸

The connection between DAMPs and autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, and others, has been established.⁸ However, the role of DAMPs in the pathogenesis of autoimmune thyroid disease and Graves' ophthalmopathy is not well understood. The immunological pathways involved in the progression of Graves' disease and the onset of Graves' ophthalmopathy, as well as the high incidence of Graves' ophthalmopathy in patients who experience remission, suggest that DAMPs may play a crucial role in the development of severe Graves' ophthalmopathy.⁹

Studies in individuals with autoimmune thyroid disease showed elevated levels of HMGB1, which correlated with thyroid antibody levels in the body.¹⁰ Lacheta et al. investigated the impact of DAMPs on inflammation in Graves' ophthalmopathy by conducting an orbital tissue biopsy to assess HMGB1 expression in orbital fatty tissue. They found an increase in HMGB1 expression that corresponded to the severity of Graves' ophthalmopathy.¹¹

Methods

This cross-sectional study compared two groups to assess the difference in blood circulation levels and the effect of HMGB1 in Graves' ophthalmopathy. Graves' Disease patients with ophthalmopathy comprised the case group, while Graves' Disease patients without ophthalmopathy comprised the control group.

Participants were identified through medical records and assessed for eligibility. Eligible participants were scheduled for a screening visit where they provided informed consent and underwent a medical history, physical examination, and Thyrotropin Receptor Antibody (TRAb) level measurement. To be eligible, participants had to have been diagnosed with Graves' disease within the past six months, be between the ages of 18 and 60, and have a Thyrotropin Receptor Antibody (TRAb) level greater than 1.75 IU/L. These criteria suggest that the study was focused on individuals who were in the early stages of Graves' disease and had elevated levels of TRAb, which is a biomarker associated with the disease. Those who met eligibility criteria were enrolled and underwent baseline assessments including laboratory tests and questionnaires. Exclusion criteria included chronic comorbidities such as diabetes, heart failure, stroke, kidney failure, cancer, thyroid ablation/surgery, other eye conditions, and active autoimmune diseases to ensure participant safety and study validity.

In 1995, the Bartley and Gorman criteria were developed to diagnose ophthalmopathy. The criteria are composed of three main components: eyelid retraction, thyroid dysfunction, and specific eye-related symptoms. Eyelid retraction is characterized by the abnormal elevation of the upper eyelid. The second criterion, thyroid dysfunction, refers to the improper functioning of the thyroid gland, which can cause a variety of symptoms, such as weight gain or loss, fatigue, and temperature sensitivity. The third criterion involves the presence of eye-related symptoms, such as optic nerve dysfunction, exophthalmos, or extra-ocular muscle involvement. Optic nerve dysfunction can cause visual disturbances, while exophthalmos is a condition in which the eyes protrude from the sockets, resulting in a “bulging” appearance. Lastly, extra-ocular muscle involvement can lead to double vision or difficulty focusing due to weakened or dysfunctional eye muscles.¹²

Circulating HMGB1 levels were measured using Enzyme-Linked Immunosorbent Assay (ELISA) method. A plasma blood sample was collected from the patients and analyzed using the HMGB1 express ELISA Kit from TECAN IBL International in Hamburg, Germany.

This study took place from September 2021 to February 2022 at RSUP Dr. Sardjito, in the Internal Medicine Clinic of Endocrinology Division, the Eye Clinic of the Oculoplasty Reconstruction Division, and the Oncology.

This study was previously approved by the subjects with written informed consent. The ethical committee of the Faculty of Medicine, Public Health, and Nursing at Universitas Gadjah Mada in Yogyakarta, Indonesia reviewed and approved this study under protocol number KE/FK/0980/EC/2021. The research adheres to the 2013 version of the Declaration of Helsinki, which outlines ethical principles for medical research involving human subjects.

Results

Differences in circulation level of HMGB1 between groups of Graves' disease with ophthalmopathy compared no ophthalmopathy

HMGB1 levels were significantly higher in Graves' patients with ophthalmopathy compared to those without, with a median value of 15.49 pg/mL in the ophthalmopathy group and 2.33 pg/mL in the non-ophthalmopathy group (p0.001). The lowest and highest values were 5.12 pg/mL to 47.59 pg/mL in the ophthalmopathy group and 0.82 pg/mL to 15.66 pg/mL in the non-ophthalmopathy group.

According to the findings of this study, the levels of HMGB1 in the blood circulation were found to be significantly different between Graves' disease patients with ophthalmopathy and those without ophthalmopathy (Figure 1).

Figure 1. HMGB1 level in patients with Graves’ Opthalmopathy and without Graves’ Opthalmopaty.

Before the regression analysis was performed, it was important to identify an appropriate cut-off value for the independent variable (HMGB1 level) which would be evaluated. This value was determined through analysis of the Receiver Operating Characteristic (ROC) curve, which aimed to find a cut-off value that had a high degree of both sensitivity and specificity. The results of the ROC analysis revealed a cut-off value of 8.86 pg/mL, with a sensitivity of 81% and a specificity of 73%.

Binary logistic regression analysis was then conducted to determine the impact of HMGB1 levels in circulation on the incidence of Graves' ophthalmopathy. The results of this analysis, showed that patients with Graves' disease and HMGB1 levels greater than 8.86 pg/mL had a 12 times higher risk of developing ophthalmopathy compared to those with HMGB1 levels less than 8.86 pg/mL (Table 2).

Table 2. Univariate and multivariate linear regression analysis: standardized Beta coefficients of independent variables and Grave disease ophthalmopath status (n=44).

Variable	Univariate				Multivariate
	N	OR1	95% CI1	p-value	OR1	95% CI1	p-value
TRAb (IU/L)	44	0.69	0.50, 0.88	0.008	0.78	0.55, 1.03	0.11
HMG Box 1 (>88.6 pg/ml))	44	12	3.11, 56.8	<0.001	7.06	1.61, 36.1	0.012
Age (Years)	44	0.99	0.90, 1.09	>0.9
Gender (Male)	44	1.32	0.30, 6.15	0.7
Smoking (Yes)	44	1	0.17, 6.01	>0.9
Free T-4 (ng/dL)	44	0.73	0.39, 1.30	0.3
Diabetes Mellitus (Yes)	44	1	0.11, 9.01	>0.9
Hypertension (Yes)	44	3.32	0.39, 70.2	0.3
Family History (Yes)	44	1.41	0.27, 7.99	0.7
Dermopathy (Yes)	44	1	0.11, 9.01	>0.9
Duration since diagnosis (months)	44	0.92	0.68, 1.24	0.6

1 OR = Odds Ratio, CI = Confidence Interval.

A stepwise analysis was conducted to determine which variables had the greatest role as predictors of the onset of Grave's disease ophthalmopathy. The results showed that high levels of TRab and HMGB1 were the variables with the greatest impact on the status of ophthalmopathy in Grave's patients. Multivariate analysis showed that even when controlling for TRab, there was still a significant statistical relationship between Grave’s ophthalmopathy and HMGB1. Table 2 showed that a high level of Hmgbox1 can increase the risk of developing Grave's ophthalmopathy by 7.06 times with a p-value of 0.012 (95% CI 1.61-36.1)

Discussion

In the study, a significant difference was observed in the levels of HMGB1 between patients with Graves' disease and ophthalmopathy and those with Graves' disease without ophthalmopathy. The median levels of HMGB1 in the group with ophthalmopathy were considerably higher compared to the group without ophthalmopathy, with a median value of 15.49 pg/mL and 2.33 pg/mL, respectively. The results of the ROC curve analysis suggested that a cut-off value of 8.86 pg/mL for HMGB1 levels had a high degree of sensitivity and specificity in predicting the presence of ophthalmopathy in patients with Graves' disease. Binary logistic regression analysis revealed that patients with Graves' disease who had elevated levels of HMGB1 (above 8.86 pg/mL) had a risk of developing ophthalmopathy that was 12 times higher compared to patients with lower levels of HMGB1. These findings align with the previous study conducted by Han et al. that concluded that patients with symptomatic Graves had higher levels of HMGB1 compared to those with stable Graves or healthy individuals.¹³

The study results showed a significant difference in the levels of HMGB1 in the circulation of Graves' disease patients with and without ophthalmopathy. HMGB1, a known effector molecule involved in inflammation, is believed to play a role in the development of Graves' disease and its associated complications, including ophthalmopathy. Its high levels have been linked with other autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, where increased HMGB1 levels and increased numbers of HMGB1-producing cells are found in inflammatory areas. Through its interaction with RAGE and TLR receptors, HMGB1 contributes to the pathogenic inflammatory processes. The study findings suggest that HMGB1 may be a useful biomarker to reflect the level of inflammation in Graves' disease patients, especially those with active disease, as increased HMGB1 secretion was found in response to high levels of pro-inflammatory cytokines such as IL-1 or TNF.¹³

The presence of elevated levels of HMGB1 in patients with Graves' disease and ophthalmopathy suggests a potential role of HMGB1 in the development of ophthalmopathy. HMGB1 is known as a Damage-Associated Molecular Pattern (DAMP) molecule and is released by damaged or necrotic cells, or cells that die without undergoing apoptosis. This provides a theoretical explanation for the high levels of HMGB1 found in patients with Graves' disease and ophthalmopathy.¹⁴

Damage-associated molecular patterns (DAMPs) play a dual role in the body as both intracellular regulators and extracellular signals of cell damage. They are typically not recognized by the immune system as long as they remain inside the cell, but are detected when they are released into the extracellular environment and trigger an immune response.¹⁴

The role of damage-associated molecular patterns (DAMPs) in the initiation of inflammation is essential for the survival of an organism. When cells are damaged or dying, DAMPs are released, acting as alarm signals to surrounding cells and activating the immune system. This process is known as the “danger signal hypothesis”.¹⁵ The immune system's response to DAMPs includes the activation of both the innate and adaptive immunity, where innate immunity acts as the first line of defense and adaptive immunity provides a more specific and prolonged response. DAMPs activate immune cells, such as macrophages and neutrophils, which phagocytose cellular debris and release cytokines and chemokines, leading to an increase in blood flow and immune cell recruitment to the site of injury. This process helps to remove damaged tissue and promote tissue repair and healing.¹³ Additionally, the presence of DAMPs can also aid in the formation of immunological memory, helping the immune system to respond more effectively in the future to similar threats.¹⁵ Overall, DAMPs play a crucial role in the body's defense mechanisms against cell damage and contribute to the regulation of tissue repair and healing.

The findings of Han et al. showed a strong correlation between the high expression of HMGB1 and its receptors with the inflammatory processes in Graves' disease with ophthalmopathy. Their research also indicated that the high expression of receptors in orbital fibroblast tissues can lead to excessive inflammation, which contributes to the growth and development of ophthalmopathy symptoms such as angiogenesis and changes in the connective and adipose tissue. Based on these findings, it is proposed that the HMGB1 signalling pathway can be targeted and inhibited through its receptors as a potential treatment option for reducing the symptoms of ophthalmopathy in patients with Graves' disease.¹³

The study by Peng et al. demonstrated that the levels of HMGB1 and RAGE were elevated in monocytes collected from patients with autoimmune thyroid disease, including Graves' disease, compared to monocytes obtained from healthy individuals. This indicates a crucial role of RAGE and HMGB1 in the development and progression of Graves' disease.¹⁶

Conclusion

The results of the study indicate a strong correlation between elevated levels of circulating HMGB1 and the occurrence of Graves' ophthalmopathy. The findings offer new perspectives on the management and treatment of ocular disease in Graves' patients by administering anti-HMGB1. By reducing HMGB1 levels, the study suggests that it may be possible to prevent the development of ocular disease and control its clinical activity in patients diagnosed early on. These results provide important implications for the clinical management of Graves' disease patients with ocular manifestations.