Oxidative stress enhances the immune response to oxidatively modified catalase enzyme in patients with Graves’ disease

Abstract Background Oxidative stress is associated with several autoimmune disorders and oxidative modification of proteins that may result in autoimmune response. This study aims to evaluate the catalase (CAT) activity and the autoimmune response against the native CAT and the oxidatively modified enzyme in patients with Graves' disease (GD) and healthy controls in a comparative way. Methods The CAT activity was evaluated via spectrophotometric method. Using enzyme‐linked immunosorbent assay, the reactivities of autoantibody toward native, malondialdehyde (MDA) and hydrogen peroxide (H2O2) modified CAT were evaluated in plasmas of patients and controls. Results Reduced CAT activity was found in patients compared with controls (P < .05). It was proved that levels of IgG antibodies against MDA‐modified CAT were higher than against unmodified ones (P < .001). No changes were found for the reactivities to H2O2‐modified CAT. Positive correlation was found between the reactivity to MDA‐modified CAT and the triiodothyronine level (P < .001, r = .6). Conclusion Our findings incriminate the MDA in the autoantibodies reactivity to oxidatively modified CAT leading to a disturbed oxidative profile and/or the progression of GD pathology.

(SOD), catalase (CAT), and glutathione peroxidase (GPx). However, when the level of ROS exceeds the defense mechanisms, these enzymes may be itself oxidatively damaged. 12 Indeed, anti-CAT autoantibodies' production was described in human autoimmune pathologies such as systemic lupus erythematosus (SLE) and arthritis rheumatoid (AR). 13 However, there is no report about the oxidative effects of ROS on CAT enzyme in thyroid disorders.
In this study, we determined whether oxidative modification of the CAT induced by MDA and H 2 O 2 enhanced the reactivity of plasmas of patients with GD and healthy controls by comparison with the reactivity toward the native enzyme.

| Ethics approval
The performed experimental protocols and consent forms were in accordance with the guidelines of the Declaration of Helsinki and approved by the local Ethics Committee of the Hedi Chaker University Hospital of Sfax, Tunisia.

| Patients recruitment
A total of 34 untreated Tunisian patients with GD were recruited from the department of endocrinology inpatient and outpatient clinics, Hedi Chaker University Hospital of Sfax, Tunisia.
In total, 65 healthy volunteers without familial history of autoimmune diseases and without any recent drug treatments were enrolled in the study (Tables 1 and 2).

| Measurement of TSH
The evaluation of the TSH level was assessed by enzyme immunoassay using the ELFA technique (enzyme-linked fluorescent assay). VIDAS TSH is an automated assay for the VIDAS system, which enables human TSH in serum or plasma to be quantitatively measured. The assay principle combines the indirect enzyme immunoassay method with a final fluorescent detection (ELFA). All reaction steps are performed by the VIDAS instrument. The disposable Solid Phase Receptacle serves both as a solid phase and as a pipetting device during the assay. Reagents for the assay are all contained in the sealed reagent strips. The results are expressed in µUI/mL.

| Measurement of FT3
The determination of the serum level of FT3 was carried out by a radioimmunoassay method using the RIA-gnost®FT3 kit (CIS bio).
This assay is based on a competition between a radioisotope-labeled antigen and a cold antigen to be assayed for the same amount of antibody. At the bottom of each tube coated with Ac, 50 µL of sample and 1000 µL of the 125I-FT3 tracer solution were successively deposited. After stirring for 120 minutes at room temperature and at a speed between 1120 and 3400 g, the liquid is removed by suction followed by taping on absorbent paper. The measurement of the radioactivity is carried out for one minute using a gamma counter (Wallac). For each series of samples, a calibration curve is established using standard solutions delivered with the kit. The results are expressed in pg/mL.

| Measurement of FT4
The RIA-gnost® FT4 kit is used to evaluate free T4 using antibody-coated tubes in a two-step procedure. The serum sample is first incubated with a polyclonal antibody bound to the solid phase and then removed by decantation. Then, the radioactive tracer is added to lodge the sites of antibodies remaining free after decantation.
At the bottom of each tube coated with Ac, 100 μL of sample and 1 mL of incubation buffer are deposited successively. After stirring for 30 minutes using a horizontal stirrer at room temperature and at a speed between 1120 and 3400 g, the liquid is removed by suction followed by tapping on absorbent paper. Then, 1 mL of the 125I-FT4 tracer solution is deposited and the tubes are stirred again for 60 minutes. After removing the remaining liquid that can adhere to the edge of the tubes by tapping, the measurement of radioactivity is carried out for one minute using a gamma counter (Wallac Anti-R-TSH (µUI/mL) <2 37 ± 2.9 Note: Statistical analyses were performed using unpaired t test; there were no significant differences between the groups regarding the age (P > .05); and significant differences were found ***P < .001.

TA B L E 2 Catalase activity in plasmas of Graves' disease patients
Test Controls n = 65

Note:
The results are expressed as mean ± SEM Statistical analyses were performed using the unpaired t test, and significant differences were found *P < .05 by comparison with the control group.
was established using standard solutions delivered with the kit.
The results were expressed in pg/mL.

| Measurement of anti-Tg and anti-TPO
The

| Catalase activity
The method of Aebi 1984 was used to evaluate the CAT activity. 14

| Enzyme-linked immunosorbent assay (ELISA)
The CAT modification was assessed according to the method of MSEDDI et al. 2017. 7 The optical density (OD) at 405 nm was determined using a micro-ELISA plate reader.

| Protein quantification
Protein Assay Kit from Bio-Rad (France) was used to determine the protein level in the plasmas of patients and controls. The bovine serum albumin served as a standard.
The plasmas are diluted in a volume of 800 µL of sterile water and then added with 200 µL of Bradford reagent. The optical density (OD) is read at 595 nm.

| Statistical analyses
The evaluation of differences between patients and controls regarding the plasmatic CAT activity and the MDA level was performed using the unpaired t test. The differences in the plasmatic reactivity to the native CAT between patients and healthy controls were assessed by ANOVA test. The evaluation of differences in plasmatic reactivities toward native and modified CAT was considered by the paired t test. The correlation study was assessed using Pearson correlation test. All data are presented as means ± standard error mean (SEM). Statistical analysis and figures were performed using GraphPad Prism 6 program.

| Evaluation of hormonal and antibodies levels
The evaluation of the level of the thyroid hormones in GD patients' showed an increase in FT4 and FT3 concomitant with reduced TSH in comparison with healthy controls (P < .001) ( Table 1). In addition, high rates of anti-thyroid autoantibodies, anti-TPO, anti-Tg, and anti-TSH-R, were detected in patients compared with controls (Table 1).

| Determination of the catalase activity
The plasmatic CAT activity was significantly reduced in patients compared with controls (P < .05) ( Table 2).

| Immune response to MDA and H 2 O 2 modified catalase
The plasmatic reactivity toward MDA-modified CAT was higher than the native one in patients with GD (P < .001) (Figure 1). Remarkably, a slight increase in the reactivity toward MDA-modified CAT was also noted in controls (P < .05) (Figure 1). Nevertheless, the ratio

| Correlation study
The statistical analyses showed a significantly positive correlation between the reactivity toward the MDA-modified CAT and the level of FT3 hormone (r = .6, P < .001, n = 31) (Figure 4).

| D ISCUSS I ON
Results reported in the present study demonstrate that the CAT activity was reduced in plasmas of patients with GD compared to controls. These data are consistent with several studies also showing a decrease in the CAT activity in sera and plasma of patients with GD. 16,17 So, the reduced activity of the enzyme may indicate an excessive consumption or structural alterations due to oxidative stress.
In fact, oxidative modifications may alter the function of the enzyme and/or create new epitopes that could generate antibody response.
Several studies supporting this hypothesis showed that the CAT enzyme is a major protein target of the lipid peroxidation product 4-HNE in autoimmune diseases such as the SLE. 18 In order to verify our hypothesis, we evaluate in a comparative way the reactivity of