2023 Volume 70 Issue 12 Pages 1169-1174
Autoimmune thyroid diseases (AITDs), such as Graves’ disease (GD) and Hashimoto’s disease (HD), are organ-specific autoimmune diseases. Histone acetylation, especially that of histone H3, is an epigenetic mechanism that regulates gene expression and is associated with the development of autoimmune diseases. However, physiological variations in histone acetylation are not yet clear, and we believe that physiological variations should be examined prior to analysis of the role of histone H3 in the pathogenesis of AITDs. In this study, we analyzed histone H3 acetylation levels in peripheral blood mononuclear cells (PBMCs) using a histone H3 total acetylation detection fast kit. Blood samples were collected before meals, between 8:30–9:00 am, daily for 10 weeks to evaluate the daily variation. At 4 days, blood was also collected before meals three times a day (at 8:30–9:00, 12:30–13:00, and 16:30–17:00) to evaluate circadian variation. Then, histone H3 acetylation levels were evaluated in AITD patients to clarify the association with the pathogenesis of AITD. Although we could not find a common pattern of circadian variance, we observed daily variation in histone H3 acetylation levels, and their coefficient of variances (CVs) were approximately 48.3%. Then, we found that histone H3 acetylation levels were significantly lower in GD and HD patients than in control subjects and these differences were larger than the daily variation in histone acetylation. In conclusion, histone H3 acetylation levels were associated with the development of AITD, even allowing for daily variation.
AUTOIMMUNE THYROID DISEASES (AITDs), such as Graves’ disease (GD) and Hashimoto’s disease (HD), are organ-specific autoimmune diseases [1, 2]. Antibodies against the thyroid-stimulating hormone (TSH) receptor are ultimately responsible for hyperthyroidism in GD. In contrast, chronic inflammation caused by autoimmune reactions destroys the thyroid tissue, resulting in HD. The intractability of GD and the severity of HD vary among patients. Some patients with GD achieve remission through medical treatment, whereas others do not. Some patients with HD develop hypothyroidism earlier in life, while some maintain a euthyroid state even up to old age. However, the intractability of GD and the severity of HD are very difficult to predict at the first diagnosis. We have already examined the associations between gene polymorphisms and various pathologic conditions of AITD to predict the development and prognosis of AITD by genetic factors and showed the significance of several gene polymorphisms [3-6]. However, autoimmune diseases, including AITD are believed to develop due to both genetic and environmental factors. In a twin study, it was recently reported that the heritability of AITD development is approximately 70–80% and that environmental factors contribute to the pathogenesis of AITD by approximately 20–30% [7]. Therefore, it is also important to clarify environmental influences to more strictly predict the development and prognosis of AITD.
Histone acetylation is an epigenetic mechanism that regulates gene expression and is an environmental factor [8]. Acetylation of core histones, especially type H3, may be involved in the regulation of chromatin structure and function, transcriptional activity and the ability of genes to bind growth factors or other promoters [9]. Thus, it can be expected that changes in histone acetylation may alter the cell proliferation/apoptosis ratio and cell cycle regulation [9, 10]. An imbalance in cell cycle regulation may initiate tumor development or progression, and several studies have reported an association of altered histone acetylation with some diseases such as cancer and autoimmune diseases [11]. Global histone H3 acetylation levels were associated with cytokine expression in peripheral blood [12, 13].
These findings suggest that alterations in histone acetylation may affect the development and prognosis of AITD by suppressing the expression of apoptosis-related genes. However, physiological variations in histone acetylation are not clear, even though they should be examined prior to analysis to ensure the significance of the changes. In this study, to clarify the physiological changes in healthy subjects, we first analyzed the circadian and daily variation in the total histone H3 acetylation level. Then, we also analyzed the histone H3 acetylation level to clarify the association of acetylation of histone H3 with the pathogenesis of AITD.
We analyzed the daily variation in histone H3 acetylation levels in 13 healthy controls, including 6 males (age 23.2 ± 2.5 years) and 7 females (age 23.3 ± 1.4 years). We excluded some samples on the day when the subjects showed clinical evidence of inflammation with fever. We also analyzed circadian variation in three healthy controls (age 22.3 ± 0.6 years).
To evaluate the daily variation in histone H3 acetylation levels, we collected a blood sample before meals between 8:30–9:00 am 1 day per week for 10 weeks. To evaluate the circadian rhythm of histone H3 acetylation levels, blood was also collected before meals three times a day (at 8:30–9:00, 12:30–13:00, and 16:30–17:00), 1 day per week for 4 weeks. The histone H3 acetylation level of samples from the same person was measured on the same plate to assess intraindividual variation. Written informed consent was obtained from all subjects, and the study protocol was approved by the Ethics Committee of Osaka University.
Subjects for analyzing the association of histone H3 acetylation with diseaseWe examined 14 patients with HD (3 males and 11 females; age 59.9 ± 15.6 years) who were positive for anti-thyroid microsomal antibody (McAb) and/or anti-thyroglobulin antibody (TgAb), 17 patients with GD (2 males and 15 females; age 59.1 ± 11.6 years) who had a clinical history of thyrotoxicosis with a positive test for anti-thyrotrophin receptor antibody (TRAb), and 9 healthy volunteers (2 males and 7 females; age 57.1 ± 12.7 years) who were euthyroid and negative for thyroid autoantibodies (control subjects). Among GD patients, 10 patients (1 male and 9 females; age 61.6 ± 9.8 years) had been treated with methimazole for at least 5 years and were still positive for TRAb (intractable GD), and 7 patients (1 male and 6 females; age 55.4 ± 13.8 years) had maintained a euthyroid state and were negative for TRAb for more than 2 years without medication (GD in remission). Among HD patients, 10 patients (3 males and 7 females; age 58.8 ± 13.8 years) developed hypothyroidism before 50 years of age and were treated with thyroxine (severe HD), and 4 patients (4 females; age 62.8 ± 14.2 years) were euthyroid and untreated over 50 years of age (mild HD). Peripheral blood was obtained between 9:00 and 12:00 to avoid the effect of circadian variation. All patients and control subjects were Japanese and were unrelated to each other. Written informed consent was obtained from all patients and controls, and the study protocol was approved by the Ethics Committee of Osaka University. The clinical characteristics of the subjects are shown in Table 1. AITD patients who were treated for other autoimmune diseases were excluded from this study.
Clinical characteristics of GD and HD patients and control subjects measured the acetylH3 levels at the sampling time
| GD | HD | Control | |||||
|---|---|---|---|---|---|---|---|
| All | Intractable | In remission | All | Severe | Mild | ||
| n | 17 | 10 | 7 | 14 | 10 | 4 | 9 |
| gender (male/female) | 2/15 | 1/9 | 1/6 | 3/11 | 3/7 | 0/4 | 2/7 |
| age | 59.1 ± 11.6 | 61.6 ± 9.8 | 55.4 ± 14 | 59.9 ± 16 | 58.8 ± 14 | 62.8 ± 14 | 57.1 ± 13 |
| Goiter size (cm) | 3.03 ± 2.5 | 3.3 ± 2.9 | 2.55 ± 2 | 2.55 ± 2.5 | 2.33 ± 2.1 | 3.3 ± 4 | — |
| FreeT4 (ng/dL) | 1.21 ± 0.22 | 1.25 ± 0.3 | 1.15 ± 0.2 | 1.4 ± 0.2 | 1.5 ± 0.2 | 1.06 ± 0.1 | — |
| TSH (μIU/mL) | 2.2 ± 1.85 | 2.48 ± 2.5 | 2.72 ± 2.1 | 1.91 ± 1.6 | 1.47 ± 1 | 3.35 ± 2.7 | — |
| TRAb (IU/L) | 9.66 ± 14.8 | 10.70 ± 15 | Negative | Negative | Negative | Negative | — |
| Current doses of anti-thyroid drug (mg/day)a | 17.1 ± 11 | 17.1 ± 11 | None | None | None | None | None |
| Current doses of L-thyroxine drug (μg/day) | 78.6 ± 26.7 | 93.75 ± 13 | 58.3 ± 29 | 76.3 ± 43 | 88 ± 42 | 37 ± 13 | None |
Values represent mean ± SD
a Doses are expressed as the comparable dose of MMI (50 mg of PTU was conveted to 5 mg of MMI)
In brief, PBMCs were isolated from EDTA-treated peripheral blood by density gradient centrifugation with Lymphoprep (density 1.077, Nycomed Pharma AS, Oslo, Norway) at 400 × g for 30 minutes at room temperature and washed in phosphate buffered saline (PBS). PBMCs were subsequently used for total histone extraction.
Total histone extraction and protein concentration quantificationTotal histones were extracted from PBMCs using a histone extraction kit (Abcam, Tokyo, Japan), according to the manufacturer’s instructions. Pre lysis buffer (1×) was added to cell debris (1 mL/L × 107 cells), and the cells were lysed on ice for 10 minutes with gentle stirring. The cells were centrifuged at 10,000 rpm for 1 min at 4°C, and the supernatant was removed. Three volumes of lysis buffer were added and incubated on ice for 30 minutes. After incubation, the cells were centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was transferred to a 1.5 mL vial, and 0.3 volumes of Balance-DTT Buffer were added to the supernatant (ex: add 0.3 mL of Balance-DTT Buffer to 1 mL of supernatant). Finally, the histone protein concentration was measured by a GE Healthcare NanoVue Plus spectrophotometer, and the extract was aliquoted and stored at –80°C.
Evaluation of histone H3 acetylation levelsWe evaluated histone H3 acetylation levels using a histone H3 total acetylation detection fast kit (Abcam, Tokyo, Japan), following the manufacturer’s recommendations. Briefly, histone proteins (0.1–1 μg) were added to the strip wells. Acetylated histone H3 was detected with a high-affinity antibody, and the levels of histone H3 acetylation (ng/mg protein) {[O.D. (sample – blank)/Slope × Protein (μg)] × 1,000} were displayed with a horseradish peroxidase-conjugated secondary antibody color development system. Color was measured by absorbance at 450 nm.
Statistical analysisWe used a paired t test between any two groups to analyze the difference in circadian rhythm and daily variation in histone H3 acetylation level. We used the Steel test to analyze the difference in histone H3 acetylation levels among the subject groups. Data were analyzed with JMP15 Pro software (SAS Institute Inc., Tokyo, Japan). P values of less than 0.05 were considered significant.
We found that acetylH3 levels did not follow common circadian rhythms; their levels changed randomly during the day. There was no clear difference in acetylH3 levels when we compared each level between 9:00 and 13:00, between 13:00 and 17:00 and between 9:00 and 17:00 (Fig. 1A–C).
AcetylH3 levels in each subject were examined independently for four days.
(A) Female 1, (B) female 2 and (C) male 1.
Daily variation in acetylH3 levels was shown in Fig. 2. The coefficient of variance (CV) of acetylH3 levels in each control subject over 10 weeks ranged from 10.2 to 98.8%. The mean CV in the daily variation in acetylH3 levels for 10 weeks was 48.3%.
Daily variations in acetylH3 levels in control subjects for over 10 weeks.
The acetylH3 levels were significantly lower in GD and HD patients than in control subjects (p = 0.0067 and p = 0.0077, respectively) (Fig. 3A). The acetylH3 levels were also significantly lower in mild HD patients than in control subjects (p = 0.0213) (Fig. 3B).
AcetylH3 levels in GD and HD patients and control subjects.
The horizontal lines indicate mean AcetylH3 levels.
Although the number of samples may not be large enough to examine the circadian variation with high statistical power in this study, we could not find a common pattern of circadian variance as shown in Fig. 1. We suggest that there will not be major common circadian variances. On the other hand, we considered that interassay variation would be minor because each sample from the same subjects was measured on the same assay plate, and the CVs of double measurement were almost within 10%. Therefore, the variations we observed in this study may simply represent intraindividual variation. As shown in Fig. 2, we observed that the daily variation in the histone H3 acetylation level and the CVs were approximately 48.3%. Therefore, to evaluate histone H3 acetylation levels, it is necessary to consider the daily variation, in other words, intraindividual variation. Since the CV of daily variation is 48.3%, differences over this variation will be considered clinically significant.
As shown in Fig. 3A, the histone H3 acetylation level was significantly lower in GD and HD patients than in control subjects. These differences were also significant even when two outliers of control subjects were excluded. Based on the intraindividual variation discussed above, the CVs in histone H3 acetylation levels between in the control and GD and HD groups were 71.2% and 69.4%, respectively, indicating that they are clinically significant because these CVs were larger than the intraindividual variation (CV = 48.3%). A previous study showed that acetylated histone H3 levels were significantly lower in patients with cancer and autoimmune diseases than in healthy controls, as shown by our results in AITD [11, 14, 15]. Supporting this, histone deacetylase 9 (HDAC9) expression in PBMCs, especially regulatory T (Treg) cells, obtained from AITD patients was higher than that in healthy controls [16]. We previously reported that the low genetic expression ability of Forkhead box P3 (Foxp3), cytotoxic T-lymphocyte associated antigen 4 (CTLA4) and programmed cell death-1 (PD-1), which regulate the function of Treg cells, was associated with the pathogenesis of AITDs [17-19]. The low acetylated histone H3 levels in AITD may suggest the suppression of Treg activation. Therefore, we suggest that histone acetylation may have some protective roles against autoimmune diseases including AITD.
There may be five limitations in this study. First, in circadian analysis, we could not examine histone H3 acetylation levels between 17:00 and 9:00 because we could not obtain peripheral blood at night. Therefore, the circadian variance of histone H3 acetylation in this study was not completely reflected. Second, since we examined daily variation only in control subjects, we could not clarify the possibility of some differences in daily variation between AITD patients and healthy subjects. Third, the samples in our study were PBMCs, not intrathyroidal mononuclear cells. The evaluation of histone H3 acetylation in intrathyroidal mononuclear cells may better clarify the roles of histone H3 acetylation in thyroid autoimmunity. Fourth, the inter-individual and daily variation in the histone H3 acetylation may be large. As described above, histone H3 levels could be indicator in the case of differences over 48.3%, further studies were needed to find more suitable indicators. Fifth, although we examined whole histone acetylation level in this study, histone modifications focus on specific locus of genes using ChIP technology may better to clarify the association of specific histone acetylation with the pathogenesis of AITD.
In conclusion, there was daily variation in the histone H3 acetylation level. A low histone H3 acetylation level was associated with the AITD susceptibility.
This work was supported by JSPS KAKENHI under grant numbers A19H040480 (to Mikio Watanabe), JP17H04111 (to Yoshinori Iwatani), and JP17K15774 (to Naoya Inoue).
The authors report that there are no competing interests to declare. Mikio Watanabe is a member of Endocrine Journal’s Editorial Board.
