Low serum interleukin‐38 levels in patients with Graves’ disease and Hashimoto’s thyroiditis

Abstract Background Autoimmune thyroid disease (AITD) mainly includes Graves’ disease (GD) and Hashimoto's thyroiditis (HT), which is caused by individual genetics, autoimmune dysfunction, and a variety of external environmental factors. Interleukin (IL)‐38 is involved in a wide range of autoimmune diseases, but little is known about IL‐38 expression in AITD. Methods Fifty patients with GD, 50 with HT, and 50 healthy controls (HC) were enrolled in this study. Basic information of the participants was obtained through a physical examination. Immunological data were obtained by an automatic chemiluminescence immunoanalyzer. C‐reactive protein (CRP) concentrations and the white blood cell count were measured. Serum IL‐38 levels were determined by an enzyme‐linked immunosorbent assay. Results Serum IL‐38 levels were significantly lower in the GD and HT groups than in the HC group (both p < 0.01). Serum CRP concentrations were significantly lower in the HT group than in the HC group (p < 0.05). Receiver operating characteristic curve analysis showed that the area under the curve was 0.7736 (p < 0.01) for IL‐38 and 0.7972 (p < 0.01) for IL‐38 combined with CRP in the GD group. In the HT group, the area under the curve was 0.7276 (p < 0.01) for IL‐38 and 0.7300 for IL‐38 combined with CRP (p < 0.01). Conclusions The results suggest that serum IL‐38 level is a potential new diagnostic biomarker in patients with GD and HT.


| INTRODUC TI ON
Hyperthyroidism is a group of clinical syndromes due to excessive synthesis and secretion of thyroid hormones. Hyperthyroidism manifests clinically as palpitations, sweating, increased eating and bowel movements, and weight loss caused by hypermetabolism and sympathetic hyperexcitability. Graves' disease (GD) is the leading cause of hyperthyroidism and characterized by decreased serum thyroidstimulating hormone (TSH) levels, increased serum thyroxine levels, and the persistence of TSH-receptor antibody (TRAb). 1 It has been demonstrated that GD is an autoimmune disease caused by a complex interaction between genetic and environmental factors. 2 Some studies show a 3% risk of GD in women and 0.5% in men throughout the lifespan. 3 The current treatment options for GD are mainly based on antithyroid medications and surgical intervention, but they have some limitations. Medication for GD leads to a high recurrence rate, while surgery results in the removal of thyroid tissue (with the risk of secondary hypothyroidism). Although the pathogenesis of GD is still not fully determined, some reports have shown that imbalance of cytokine expression levels in vivo plays a key role in the pathogenesis of the disease. 1 In addition, some studies show that antithyroid drugs can reduce the production of thyroid pro-inflammatory cytokines. 4 An increasing number of studies have suggested that proinflammatory cytokines, such as interleukin (IL)-35, IL-29, IL-27, and tumor necrosis factor (TNF)α, may be involved in the development of GD. [5][6][7][8] Hashimoto's thyroiditis (HT) is an important component of autoimmune thyroid disease (AITD). HT is also an important cause of hypothyroidism and pathologically characterized by diffuse parenchymal atrophy, lymphocytic infiltration, and fibrosis. 9 In addition, patients with HT are at higher risk of being affected by cardiovascular disease. 9,10 Currently, the incidence of HT is 0.3-1.5 cases per 1000 people 9 ; the affected population is predominantly female, and the incidence in men is one-tenth of that in women. 9,11 At present, there is no particularly good treatment for HT, and hypothyroidism is mainly controlled by hormone replacement (oral Levo-Thyroxine4).
Although the exact etiology of HT has not yet been fully clarified, cellular and humoral immunity plays a key role in the development of this disease. Evidence suggests that serum cytokines, such as IL-6, TNFα, IL-10, IL-17, and IL-22, play an important role in the pathogenesis of HT. [12][13][14] IL-38, which is a member of the IL-1 family, is highly homologous to IL-1 receptor antagonist (IL-1Ra) and IL-36Ra, and is functionally an anti-inflammatory cytokine. 15 IL-38 is mainly expressed in the placenta, heart, liver, thymus, spleen, and fetal tissue. 15 A significant association has been reported between IL-38 levels and autoimmune diseases, such as rheumatoid arthritis (RA), 16 systemic lupus erythematosus (SLE), 17 and Behçet's disease. 18 On the basis of the above-mentioned evidence on the role of IL-38 in autoimmune diseases and the paucity of relevant data in AITD studies, we aimed to investigate serum IL-38 expression in patients with AITD and its relationship with corresponding antibodies.

| Research subjects
The subjects in this study were divided into the GD group, HT group, and healthy control (HC) group, with 50 people in each group. All participants were outpatients or inpatients at the Affiliated Lihuili The inclusion criteria were as follows: subjects in the GD group met the diagnostic criteria of GD; subjects in the HT group met the diagnostic criteria of HT; and all participants were aged ≥18 years. The exclusion criteria were as follows: pregnancy, hypoproteinemia, hormone administration, infection, trauma, and other autoimmune diseases.

| Biochemical parameter measurements
All subjects fasted overnight before blood collection, and blood samples were collected from 8:00 to 9:00 a.m. on the following day.
Blood samples were stored at room temperature for 2 h and centrifuged at 1000 × g for 20 min, and the supernatant was collected and stored in sterile enzyme-free tubes at −80°C.
TRAb was detected using a fully automated chemiluminescent immunoassay analyzer (MAGLUMI4000plus; Shenzhen New Industries).
Biochemical parameters, such as the white blood cell count (WBC) and C-reactive protein (CRP) concentrations, were measured by an automatic biochemical analyzer (AU5800; Beckman). All measurements were carried out in strict accordance with the equipment operating procedures.

| IL-38 measurement
Enzyme-linked immunosorbent assays (ELISA) were used to measure serum IL-38 protein concentrations in all participants. These ELISA kits were obtained from Shanghai Jianglai Biotechnology Co., Ltd. The experimental procedure was performed in strict accordance with the manufacturer's instructions. All serum samples were tested in duplicate. The same numbers of serum samples from different groups were randomly selected for testing in the same analytical batch.

| Statistical analysis
Statistical analysis and creation of graphs were performed using GraphPad Prism 8.0 (GraphPad Software Inc.). Measurement data are expressed as mean ± standard deviation (SD). Count data are expressed as the percentage. For the measurement data, one-way analysis of variance was used for comparisons between the three groups, and the independent samples t test was used for comparisons between two groups.
Comparisons between the count data were performed using the chisquared test. Principal component analysis was used to assess the ability of IL-38, CRP, WBC, and thyroid-related parameters to classify all samples in the three groups. Pearson correlation analysis was performed to assess the correlation between IL-38, CRP, and thyroid-related parameters. p < 0.05 was considered to be statistically significant. Table 1 shows the basic clinical information and biochemical parameters of the participants in the HC, HT, and GD groups. The mean age of participants and the proportion of women in the HT group were significantly higher compared with those in the HC group (both p < 0.05).

| Clinical and biochemical parameter data
The remaining biochemical parameters related to AITD were consistent with the characteristic manifestations of HT and GD.

| Serum IL-38 and CRP concentrations, and the WBC count in AITD
We found that IL-38 concentrations were reduced in the HT (p < 0.01) and GD (p < 0.01) groups compared with those in the HC group ( Figure 1A). The HT group had lower CRP concentrations than those in the HC group (p < 0.05), while the GD group had higher CRP concentrations than those in the HT group (p < 0.05) ( Figure 1B).
CRP concentrations tended to be higher in the GD group than in the HC group, but this was not significant. The WBC count was not significantly different between the three groups ( Figure 1C).

| Principal component analysis
We performed principal component analysis in the three groups of participants by combining IL-38, CRP, WBC, and other parameters reflecting thyroid function. These indicators were able to separate HT and GD from HC (Figure 2A,B).

| Correlation analysis between serum IL-38 concentrations, inflammatory markers, and thyroid function parameters
We constructed a visual correlation heat map between serum IL-38 concentrations, inflammatory markers, and thyroid-related parameters ( Figure 3A,B). On the basis of the correlation heat map, Pearson cor-

| Receiver operating characteristic curves for the diagnosis of AITD
We evaluated the diagnostic value of IL-38 concentrations and IL-38 combined with CRP concentrations for AITD using receiver operating characteristic (ROC) curves ( Figure 4). The area under curve (AUC) was

| DISCUSS ION
To the best of our knowledge, this is the first study to assess the importance of serum IL-38 levels for diagnosing AITD. We found that serum IL-38 concentrations in the GD and HT groups were significantly lower compared with those in the HC group. We also found lower CRP concentrations in the HT group compared with those in the HC group.  (Table 1). Thyroid-associated ophthalmopathy (TPO) is an organ-specific autoimmune disease closely related to GD. Shi et al. 26 showed that IL-38 concentrations with TPO in vitro. 27 We also observed lower serum CRP concentrations in patients with HT compared with those in patients with HC. We found that serum CRP concentrations tended to be higher in the GD group than in the HC group. Few studies have reported the relationship between serum CRP concentrations and AITD. Rao et al. 28 showed that saliva CRP concentrations were lower in patients with HT than in the normal population, which supports our finding. CRP, which is an indicator of inflammation, is an acute phase

| CON CLUS ION
This study shows that serum IL-38 concentrations are lower in GD and HT compared with the healthy population. Additionally, serum CRP concentrations are significantly lower in HT than in the healthy population and tend to be increase in GD. Furthermore, serum IL-38 and CRP concentrations have good diagnostic value for HT and GD.
Our findings will be helpful for future mechanistic studies and for the prevention and treatment of AITD.

CO N FLI C T S O F I NTE R E S T
The authors declared that they have no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.