Evaluation of IL-29 in Euthyroid Patients with Graves' Orbitopathy: A Preliminary Study

Background The most frequent cause of hyperthyroidism is Graves' disease (GD). Orbitopathy is the most prevalent and recognizable extrathyroidal manifestation of Graves' disease with unrevealed pathogenesis. Interleukin 29 (IL-29) is a relatively newly discovered inflammatory cytokine. Thus, the aim of this study was to evaluate the relationship between IL-29 and Graves' orbitopathy (GO) in euthyroid patients. Methods Thirty-one euthyroid patients with Graves' disease and with active GO [clinical activity score (CAS) ≥ 3/7], seventeen euthyroid patients with GD but without GO, and seventy-two healthy control subjects (CS) matched for age and gender were enrolled in the study. The following parameters were evaluated in every participant: thyroid-related hormones and autoantibodies and inflammatory markers (white blood cells, hsCRP). ELISA assay was applied to measure the concentration of IL-29. Results We found higher level of IL-29 in GO group in comparison with CS [165 (133-747) vs. 62 (62-217) pg/mL, p < 0.001]. Furthermore, participants in the subgroup with GD with GO as compared with GD without GO had higher concentration of IL-29 [165 (133-747) vs. 62 (62-558) pg/mL, p = 0.031]. The ROC analysis for IL-29 revealed IL-29 cut-off of 105 pg/mL (sensitivity 1.000 and specificity 0.597) as the best value significantly indicating the presence of GO in GD [area under the ROC curve (AUC): 0.739, 95% confidence interval (CI): 0.646-0.833, p < 0.001]. Conclusions The present study revealed for the first time an elevated level of IL-29 in the serum of patients with GD and GO that might suggest its involvement in the pathogenesis of GD ocular complications.


Introduction
Graves' disease (GD) is recognized as the most common cause of hyperthyroidism, and the risk of development of GD during the whole life is estimated at 3% in women and 0.5% in men [1], and anti-TSH receptor antibodies (TRAb) play the main role in the pathogenesis of GD. Graves' orbitopathy (GO), also called ophthalmopathy, is an extrathyroidal manifestation of GD affecting the eye muscles and retroorbital fat. In European population, the prevalence of GO is estimated about 1 case per 1000 people [2]. Activity of GO can be assessed using clinical activity score (CAS), and CAS ≥ 3/7 indicates an active GO. Severity can be expressed in simple staging system as mild, moderate to severe, and sight threatening (sometimes called very severe) [3]. It is estimated that about 5% of patients with GD suffer from moderate-to-severe GO [4]. The main treatment option for GO is therapy with corticosteroids. In the last few years, some new methods were applied to treat GO: rituximab (a monoclonal antibody against the protein CD20 on the surface of B cells) [5], tocilizumab [interleukin-6 (IL-6) receptor antibodies] [6], and teprotumumab (monoclonal antibody being an inhibitor of insulin-like growth factor I receptor) [7]. They resulted from recently discovered new immune pathways which provided a basis to develop new treatment options. Unfortunately, effectiveness of mentioned drugs is limited, so new medications are still searched for.  is also recalled as interferon lambda 1 (IFN-λ1). This protein is quite a new member of the recently discovered IFN-λ family and plays a strong antiviral role [8]. Moreover, it is known that, without any exposure to viruses, dendritic cells and macrophages produce Il-29 during wide range of diseases with autoimmune aetiology [9]. The elevated levels of IL-29 were already detected in some autoimmune diseases such as Sjögren syndrome, rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, and psoriasis [10][11][12][13][14]. Moreover, elevated concentrations of IL-29 were found in atopic dermatitis and asthma [15,16]. Other interleukins, such as IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, and IL-28B, are closely related to IL-29. They together form a large family called IL-10 family [17]. We know now that polymorphisms of the genes of IL-10, IL-22, and IL-28 are associated with a higher prevalence of autoimmune thyroid disease [18][19][20]. Until the present study was conducted, only one research aimed to evaluate the role of IL-29 in thyroid disorders. In the cited study, elevated serum levels of IL-28 and IL-29 were detected in patients with Hashimoto's thyroiditis (HT) [20].
The concentration of IL-29 in Graves' orbitopathy has not been evaluated yet. The most valuable results can be drawn from the observation of IL-29 in patients with Graves' orbitopathy in euthyroidism. Thus, the aim of the present study was to assess the concentration of IL-29 in euthyroid patients with Graves' orbitopathy in comparison with the healthy controls and euthyroid patients with Graves' disease without orbitopathy. CS were examined clinically (no sign of thyroid or other inflammatory disorders), using ultrasound (US) imaging (normal result), and underwent the same laboratory tests as the study group (all results within reference interval).

Patients and Methods
All experimental protocols are in accordance with the 1975 Declaration of Helsinki and were approved by the Bioethical Committee of Poznan University of Medical Sciences (approval numbers: 174/17 and 87/19). Every participant was involved voluntarily and delivered a written informed consent to participate in the study. All data needed to reproduce the results of this study are available from the corresponding author upon reasonable request.

Graves' Disease and Graves' Orbitopathy Assessment.
Graves' disease was diagnosed if the following criteria were fulfilled: clinical symptoms of hyperthyroidism and/or characteristic US image (increased size of thyroid gland, echotexture hypoechoic or heterogeneous, and hypervascular on color Doppler) and biochemical indicators (low thyrotropin-TSH, high free triiodothyronine-FT3, high free thyroxine-FT4, and elevated anti-TSH receptor antibodies-TRAb) [21]. Mentioned information was taken from the hospital database or patients' anamnesis. Moreover, every patient underwent an ophthalmological examination of GO in accordance with the European Group on Graves' orbitopathy (EUGOGO) guidelines [22,23]. The actual severity of GO was assessed using clinical activity score (CAS), where CAS ≥ 3/7 indicated an active GO. Moreover, to confirm the diagnosis of GO, magnetic resonance imaging (MRI) of orbits was performed. Patients in our study were already treated with antithyroid medications and euthyroid, which means that TSH, FT3, and FT4 were within the normal range. Every patient with GO enrolled in our research had GD and active GO.

Statistical Analysis.
The acquired data were analysed and presented statistically using Statistica V13 (StatSoft, Tulsa, Oklahoma, USA, RRID: SCR_014213, https://www.tibco .com). At first, normality was analyzed by Shapiro-Wilk test. The majority of qualitative variables did not follow normal distribution (only distributions of FT3 and FT4 were normal), 2 Mediators of Inflammation which determined the use of nonparametric statistical tests. Qualitative parameters are expressed as median with 25-75% interquartile range (IQR). In descriptive characteristics, two groups were compared using nonparametric Mann-Whitney U test (except sex which was compared using Chi-squared test). The Spearman's rank correlation coefficients were calculated to estimate the correlations. Additionally, IL-29 was also evaluated in receiver operating characteristic (ROC) analysis with the Youden index to determine the cut-off [26]. The p value threshold was set as statistically significant in every analysis at <0.05.

Results
Studied group comprised 31 participants diagnosed with GD with GO, 17 participants with GD without GO, and the control group included 72 healthy subjects (CS). A comparison of basic parameters is provided in Table 1. CS were matched for age and gender. Compared groups did not differ according to markers of inflammation, such as CRP and WBC.  Table 2). Statistically significant positive correlation was found between IL-29 level and WBC (r = 0:45, p = 0:012) in Graves' disease with GO. Age, ALT, AST, and hsCRP did not correlate statistically significantly with concentration of IL-29 neither in patients with GD and GO nor with GD without GO.

Discussion
In the present study, we demonstrated that increased serum level of IL-29 was associated with active GO in euthyroid patients with GD. Moreover, increased serum concentration of IL-29 was found in euthyroid patients with GD and GO as compared with euthyroid patients with GD and simultaneously without GO. To the best of our knowledge, our study is the first evaluating IL-29 serum concentrations in GO. We assessed patients in the euthyroid state, which excludes the potential influence of thyroid hormones on the level of IL-29. What is more, very high sensitivity in the ROC analysis for IL-29 at GO diagnosis suggests potential of Il-29 in diagnostics. In the future, IL-29 can be a potential point of action of the novel drugs for GO. Taken together, our results might suggest the potential relationship between the thyroid autoimmunity and IL-29, which can be very useful in clinical setting.
In the discussion of the results of the present study, the most important is single report focused on IL-29 in HT conducted on 99 patients [20]. The cited study revealed increased IL-29 serum level in HT patients if compared with healthy controls. It suggests the involvement of IL-29 in the pathogenesis of autoimmune thyroid disease. As mentioned above, there are no other studies which assessed the concentration of IL-29 in Graves' disease; thus, we are unable to compare present results with any more studies.
Pathogenesis of GO is still not fully determined, which results in limited therapeutic modalities [27]. We know some inflammatory mediators which gene overexpression was found in orbits of patients with GO. In one study, eye muscles and retroorbital fat tissue were examined as they are two major sites involved in the development of GO. The   Mediators of Inflammation severity of the inflammatory process positively correlated with TNF-α mRNA and IL-6 mRNA expression and negatively with IL-4 mRNA and IL-10 mRNA expression [28]. Furthermore, some pathways were already used in clinical trials: tocilizumab [interleukin-6 (IL-6) receptor antibodies] [6] and teprotumumab (monoclonal antibody being an inhibitor of insulin-like growth factor I receptor) [7]. The results of mentioned trials are very promising. What is more, also, IL-7, IL-8, IL-15, IL-16, and IL-17 are engaged in GO pathogenesis that suggests multifactorial background [29][30][31][32].
The action of IL-29 on eye muscle and retroorbital fat tissue can occur via some signaling pathways. Potential pathways that take part in GO development include nuclear     Mediators of Inflammation factor-kappa B (NF-κB) signaling pathway [33], which is induced by IL-29 [34]. IL-29 is also an activator of Janus kinase/signal transduction and activator (JAK-STAT) through STAT1, STAT2, STAT3, and STAT5 [35]. What is essential, thyroid-associated orbitopathy is an effect of the activation of JAK-STAT [36]. Moreover, IL-29 is an inductor of mitogen-activated protein kinase (MAPK), and MAPK is possibly engaged in GD development [37,38]. IL-29 also affects B lymphocytes, which are responsible for TRAb production [39]. This might explain the correlation of TRAb and IL-29 observed in our study. Another action of IL-29 is to stimulate monocytes to excretion of IL-6, IL-8, IL-10, and IL-8 which is proved to be strongly associated with GD [40,41]. Some limitations of the present study should be mentioned. This is a preliminary study on relatively small sample size. Relatively small number of participants is a result of restrictive inclusion criteria such as full biochemical euthyroidism in every enrolled patient. Moreover, patients in the group with GD and GO were older than patients in group with GD and without GO, but age did not correlate statistically significantly with concentration of IL-29 that excludes potential bias. Furthermore, any judgments about causality between IL-29 and GO cannot be made as we had a single point of measurements. However, the strengths of the present study must be addressed too. There are strict inclusion and exclusion criteria that eliminate wide range of possible biases. Enrollment of only euthyroid subjects allowed us to examine the impact of autoimmunity without bias in the form of hormonal imbalance.
What is also essential, applied methods are minimally invasive and widely available that makes our results replicable and easy to introduce in clinical practice in the future. The present study can serve as a great background to plan a prospective study evaluating the relationship between IL-29 and GO.

Conclusions
The present study revealed for the first time an elevated level of IL-29 in the serum of patients with GD and GO that might suggest its involvement in the pathogenesis of GD ocular complications. The potential role of IL-29 as a predictor of GO occurrence needs to be confirmed in studies conducted on larger cohorts of GO patients. Moreover, future investigations are required to evaluate the clinical potential of IL-29 as a therapeutic target in GO.

Data Availability
The data used to support the findings of this study may be released upon application to the corresponding author, who can be contacted at the address Department of Endocrinology, Metabolism and Internal Medicine, Przybyszewskiego 49, 60-355, Poznan, Poland, and at the e-mail: bogusz.falkowski@onet.pl.