Abstract
Objective
A vicious cycle between circadian disruption and escalating immune responses has been described in diverse inflammatory disease. The current study aimed to explore the role of circadian clock disruption in autoimmune thyroiditis (AIT).
Methods
Thirty AIT patients and 30 controls were enrolled and biopsied for thyroid tissues. Alterations of core clock genes expression in AIT thyroid tissues, and its association with serum and tissue inflammatory biomarkers were assessed. For animal studies, C57BL/6J mice administered with porcine thyroglobulin or PBS (as control) combined with adjuvants were sacrificed at four time points to investigate the circadian characteristic of experimental autoimmune thyroiditis (EAT). Light shift (LS) conditions were used to explore the influence of external circadian disturbance on EAT.
Results
The expression of clock genes BMAL1 and PER2 was significantly reduced in thyroid tissues from AIT patients and was negatively correlated to levels of thyroid peroxidase antibodies. In mouse models, diurnal fluctuations of proinflammatory cytokines were demonstrated, and further exposing mice to LS led to overproduction of TNF-α, IFN-γ, and anti-thyroglobulin antibodies. Circadian analysis revealed significant oscillations of Bmal1, Clock, Per2, Cry1, Ror, and Rev-erb, which was broadly disturbed in EAT, LS, and EAT + LS groups.
Conclusions
This study demonstrates that expression pattern of clock genes was disrupted in AIT thyroid, and chronic circadian disruption may aggravate the inflammatory responses in AIT. Whether maintaining a regular circadian rhythm can alleviate autoimmune thyroid diseases warrants further research.
Introduction
Autoimmune thyroiditis (AIT), also referred to as Hashimoto’s thyroiditis (HT) or chronic lymphocytic thyroiditis, is a common autoimmune disorder with a prevalence of 0.3–1.5 cases per 1000 subjects per year (1). AIT is characterized by the overproduction of thyroid autoantibodies and lymphocytic infiltration within the thyroid tissue (2). In certain cases, AIT may finally evolve into hypothyroidism, generating a spectrum of clinical conditions ranging from fatigue, weight gain, depression and cardiovascular events (3). Typically, AIT starts from the loss of immune tolerance toward thyroid-specific self-antigens due to genetic predisposition, environmental factors, gut microbiome, and immunity. However, the risk factors and pathogenesis of AIT remain elusive (1).
Circadian clocks are biological pacemakers that enable organisms to adapt and anticipate their physiological behaviors, such as sleep, metabolism, and mood to predictable environmental cues. In mammals, the circadian clock is also known as the internal biological clock, which is hierarchically made up of one central clock residing in the suprachiasmatic nucleus (SCN) of the hypothalamus and multiple peripheral cellular clocks (4). The central SCN synchronizes with light–darkness cycles through the retino-hypothalamic tract receiving photic stimulus. As only the retina possesses photosensitivity in mammals, peripheral clocks are primarily entrained by the central clock through the autonomic nervous system and endocrine hormones (5). Moreover, emerging evidence suggests that peripheral clocks can also respond to external cues, including food intake, exercise, and temperature, independent of the central clock (6).
At the molecular level, both the central and peripheral clocks are genetically encoded by three basic transcription–translation feedback loops. The primary loop of the circadian clock consists of a heterodimer transcriptional complex composed of BMAL1 and CLOCK. This complex binds to the E-box elements of target gene promoters to activate transcriptional repressors PER and CRY, as well as other clock-controlled genes. The main BMAL1/CLOCK loop together with two supporting loops governed by ROR/REV-ERB and D-box transcription factors form a 24-h periodicity (7). This periodicity has been observed in various immune processes, such as lymphocyte trafficking, antigen presentation, and the production of inflammatory cytokines (8), while several human diseases including rheumatoid arthritis (RA), multiple sclerosis (MS), and psychiatric disorders can lead to circadian disruptions (9, 10, 11). In addition, the asynchrony of the internal biological clock (circadian physiological and behavioral processes over a 24-hcycle) with the environmental light–darkness cycle is one of the main mechanisms mediating the pathologies of chronic inflammatory diseases (8, 12, 13).
In recent years, the clock machinery was identified not only in multiple types of immune cells but also in structural cells that form part of the ‘circadian immune circuit’ (5). Likewise, thyroid physiology is under the precise regulation of the circadian system. Animal studies suggested that the circadian rhythms of thyroid-stimulating hormone (TSH) and thyroid hormones (T3 and T4) persist even in the absence of the SCN control, demonstrating the self-sustaining nature of the thyroid clock (14). However, only a few human studies point to a potential role of clock genes in patients with thyroid nodules or thyroid cancer (15, 16). Furthermore, the involvement of thyroid-intrinsic clock in thyroid autoimmunity has not been evaluated.
Hence, we hypothesized that there might be a reciprocal interaction between circadian disturbance and inflammation in the development of AIT. To test this hypothesis, we examined the circadian characteristics in AIT and investigated the effects of light shift (LS) on thyroid inflammation.
Materials and methods
Recruitment of patients and sample collection
All studies were in accordance with the principles set out in the Declaration of Helsinki. This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University (No. 2018-71-3). Thyroid tissues collected for this study were from the remaining biopsy specimens used for pathological examination, and the blood test data of participants were retrospectively obtained from the medical records. All participants provided informed consent that their clinical samples and medical records could be used for research purposes.
Human thyroid tissues from 30 AIT patients and 30 age- and sex-matched controls were obtained from the subjects who underwent thyroidectomy for thyroid adenomas, thyroid nodules, or goiter between January 2020 and September 2021. We did not perform a power analysis as there is a lack of information on the effect size of clock genes in thyroid tissue from prior research. The sample size of 30 patients was chosen based on practical factors, including the accessibility of eligible participants during the study period and the feasibility of collecting data. Patients with elevated serum thyroid autoantibodies (thyroid peroxidase antibodies (TPOAb) >200 IU/mL and/or anti-thyroglobulin antibodies (TgAb) >200 IU/mL) and pathological examination of AIT were recruited in the AIT group; subjects with normal thyroid autoantibody levels and pathological examination of benign thyroid nodules were recruited in the control group. The following exclusion criteria were applied to all groups: (i) thyroid cancer or other malignant disease; (ii) thyroid dysfunction, exposure to radioactive iodine treatment, antithyroid medication or thyroid hormone replacement; (iii) systemic autoimmune disease, acute inflammatory disease or pregnancy. Serum samples were obtained at 08:00 h in the morning before surgery to measure thyroid functions (TSH, free thyroxine (FT4), free triiodothyronine (FT3)) and thyroid autoantibodies (TgAb, TPOAb) (electrochemiluminescence method, Architect i2000SR, Abbott Laboratories, USA) and stored at −80°C for further analysis.
All tissues used in this study were adjacent to the thyroid nodules. The tissue specimen was placed on ice and taken to the laboratory for processing immediately after excision. Each thyroid sample was divided into aliquots, half was immersed in 4% paraformaldehyde (PFA) for paraffin embedding, and another half was frozen in liquid nitrogen and stored at −80℃ for further analysis.
Animals and study design
All animal experiments were performed via standardized protocols under anesthesia with intraperitoneal injection of pentobarbital and were ethically approved by the Department of Laboratory Animals of the First Affiliated Hospital of China Medical University (No.KT2018023).
Female C57BL/6J mice were purchased from GemPharmatech Co., Ltd (Jiangsu, China) and separated into EAT (experimental autoimmune thyroiditis, a mouse model for AIT) and NC (normal control) groups randomly. All animals were raised in a clean room with 12-h light and 12-h darkness (light dark (LD)) conditions with light on at 07:00 h (defined as Zeitgeber time 0, ZT0) and lights off at 19:00 h (defined as ZT12), with free access to food and water. Animals were housed in the same environment to minimize potential variations in estrous cycles caused by external factors. For the daytime culling, mice were exposed to a light source to ensure proper visualization during the procedure. For nighttime culling, red lights were used in the room to avoid disrupting the natural darkness cycle of the mice.
First, to explore the diurnal rhythms in AIT, 16-week-old mice in EAT and NC groups were sacrificed at ZT0, ZT6, ZT12 and ZT18 for measurement of disease severity, cytokines and circadian genes expression profiles. Secondly, given that light is the most dominant regulator of biological rhythms (17), we employed a light-induced phase shift model to examine the impacts of external clock disruption on the development of AIT and thyroid clock (Supplementary Fig. 1, see section on supplementary materials given at the end of this article).
Induction and assessment of EAT
For the induction of EAT, 100 μg porcine thyroglobulin (pTg, Jianglai Biotechnology, Shanghai, China) emulsified in 100 μL complete Freund’s adjuvant (CFA, Beyotime Biotechnology, Shanghai, China) was subcutaneously injected into 10-week-old C57 BL/6J mice on day 0. A second subcutaneous injection using the same amount of pTg emulsified in incomplete Freund’s adjuvant (IFA, Beyotime Biotechnology, Shanghai, China) was performed on day 14. As a control, animals were injected with an emulsion of PBS and CFA/IFA in equal volumes and time intervals. Mice thyroid glands collected were cut into 10-μm sections and stained with hematoxylin and eosin (H&E) for disease severity evaluation. EAT scores were obtained using a discrete scale ranging from 0 (indicating normal) to 5 (representing complete thyroid infiltration and destruction) as previously described (18, 19), and a higher EAT score indicates a more severe level of lymphocytic infiltration.
Sample collection and thyroid histopathology
Animals’ blood samples were first collected by cardiac puncture after anesthesia, and then immediately sacrificed. Serum samples were isolated from the blood samples after centrifugation (1000 g , 10 min, 4℃) and stored at −80℃ for later analysis. One thyroid gland lobe was stored at −80℃ having been frozen for 10 min in liquid nitrogen. Another thyroid gland lobe was placed in 4% PFA for 12 h, and underwent gradient dehydration in 15%, 30% sucrose for 2 days. Tissue sections were cut to 2 μm thickness. Primary antibodies against BMAL1 (NB100-2288, dilution 1:500; Novus, USA) and PER2 (NB100-125, dilution 1:250; Novus) and HRP-conjugated secondary antibodies (1:2000) supplied in a DAB kit (CW2069S, CWBIO, Beijing, China) were used for immunostaining of thyroid sections. To validate the specificity of the antibodies used for immunocytochemistry, we performed primary controls (+primary antibody/−secondary antibody) and secondary controls (−primary antibody/+secondary antibody), both of which showed no positive labeling. Immunohistochemistry images were taken with Leica Aperrio Versa 8 microscope (Leica), details of quantification by IHC Profiler (20) were listed as Supplementary Data 1.
RNA extraction and quantitative RT-PCR (q-PCR)
Total RNA from frozen thyroid tissue (10–20 mg) was extracted with Trizol Reagent (Thermo Fisher Scientific) and quantified with NanoDrop 2000 (Thermo Fisher Scientific). The purity of RNA samples was assessed based on the A260/A280 ratio, and samples with an A260/A280 ratio ranging from 1.8 to 2.0 were selected for further experiments. First-strand cDNA was synthesized from 2.5 μg of total RNA using the PrimeScript RT reagent Kit (RR036A, Takara Bio) in a total volume of 20 µL. For quantitative PCR analyses, 50 ng cDNA were used as templates after quantified with the NanoDrop 2000, 20 μL reaction mixtures were prepared using SYBR-Green (RR820A, Takara Bio).
The primers used for the analyses are shown in Supplementary Table 1. Quality control of q-PCR experiments is presented in Supplementary Data 2. Relative mRNA levels were normalized against Gapdh using the 2−ΔΔCt method.
Cytometric bead array (CBA) analysis
Serum levels of IL-1β, CXCL10, IL-7, IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α and IFN-γ were detected by cytometric bead array, and the data were obtained by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Specifically, the serum samples were incubated with capture beads coated with specific antibodies against the cytokines of interest, followed by the detection with fluorescently labeled detection antibodies. The fluorescence signal of the beads was then analyzed by flow cytometry to quantify the concentration of each cytokine.
Enzyme-linked immunosorbent assay
Quantification of human serum melatonin, mouse serum TgAb, cytokine (TNF-α, IL-17, IL-6, IL-10, IFN-γ, transforming growth factor (TGF)-β) and cortisol levels was performed with ELISA kits (Meimian, Jiangsu, China) as per manual and analyzed under a wavelength of 450 nm.
Statistical analysis
Data are presented as the median (interquartile range, IQR) or mean ± standard error of the mean (s.e.m.) from at least three independent experiments. Statistical analysis was performed using SPSS version 26 (IBM). Mann–Whitney U test was used to analyze differences between the two groups. Two-way ANOVA was performed to determine the effect of treatment factors (AIT and LS), time and their interaction effects on the expression of clock genes. One-way ANOVA and cosinor analysis were performed to ascertain circadian expression of clock genes. Acrophase and relative amplitude for circadian genes were estimated for circadian gene-sets using Cosinor within Discorhythm (version 1.2.1) (21). Linear regression analysis was performed to evaluate the association between mRNA expression of core clock genes and inflammatory biomarkers. P values < 0.05 were perceived as statistically significant. Graphics were created using Prism version 9.0 (GraphPad Software).
Results
Altered thyroid clock gene expression in AIT patients
The clinical characteristics of the subjects are shown in Table 1, and time of thyroid tissue sample collection is provided in Supplementary Table 3. No significant differences in serum FT3, FT4, and TSH concentrations were found between AIT and control group, while levels of TPOAb and TgAb were significantly elevated in the AIT group (all P < 0.001). Furthermore, serum levels of melatonin, a parameter reflecting the regulation of central clock SCN (22), were not significantly different between AIT and control group.
Clinical characteristics of participants.
| AIT | Control | P | |
|---|---|---|---|
| Age (years) | 47 (35–55) | 48 (35–56) | 0.844 |
| Sex (females, %) | 25, 83.3% | 24, 82.8% | 0.953 |
| BMI (kg/m2) | 24.97 (22.85–26.91) | 24.65 (21.74–26.50) | 0.306 |
| TSH (mIU/L) | 1.83 (1.14–3.33) | 1.35 (1.03–2.03) | 0.095 |
| FT3 (pmol/L) | 4.33 (4.07–4.62) | 4.58 (4.15–4.76) | 0.252 |
| FT4 (pmol/L) | 12.64 (11.39–13.33) | 13.44 (12.11–14.24) | 0.081 |
| TPOAb (mIU/L) | 227.98 (79.55–420.26) | 0.99 (0.61–1.58) | <0.001 |
| TgAb (mIU/L) | 215.40 (38.34–632.53) | 1.36 (0.82–2.57) | <0.001 |
| Melatonin (pg/mL) | 26.59 (19.86–35.46) | 23.35 (18.81–31.92) | 0.331 |
The figure in brackets represents the interquartile range (IQR) of the variable.
FT3,: free triiodothyronine; FT4, :free thyroxine; TgAb, thyroglobulin antibodies; TPOAb, thyroid peroxidase antibodies; TSH, thyroid-stimulating hormone.
To characterize alterations of thyroid clock in AIT, we first used q-PCR to detect transcripts of six core clock genes (BMAL1, CLOCK, PER2, CRY1, REV-ERB and ROR) in thyroid samples. The mRNA levels of BMAL1 and PER2 were significantly reduced in AIT thyroid compared with control group (Fig. 1A). Consistently, IHC analysis demonstrated significantly reduced BMAL1 and PER2 in AIT thyroid (Fig. 1B and C), indicating local thyroid clock defects in AIT. Cosinor analysis was performed to account for the variations of sample collection time, and the results did not reveal a significant circadian trend of clock gene expression (Supplementary Table 4).
Altered core clock gene expression in patients with AIT. (A) Comparisons of relative mRNA levels of core clock genes in thyroid tissues of patients with AIT and control (n = 25–30/group). (B) Representative IHC sections of BMAL1 and PER2 for thyroid samples from the same AIT and control subject (counterstained with Hematoxylin), yellow arrow indicates the positive staining area (magnification = ×400; scale bar = 50 μm). (C) Quantification of BMAL1 and PER2 by IHC intensity (n = 30 per group). Data represent the mean ± s.e.m.*P < 0.05; **P < 0.01;***P < 0.001; ns, not significant; two-way significance was calculated by Mann–Whitney U test.
Citation: European Thyroid Journal 12, 5; 10.1530/ETJ-23-0035
Secondly, we evaluated the inflammatory biomarkers reflecting systemic and thyroid inflammation in subjects and analyzed their association with mRNA levels of BMAL1 and PER2. As expected, AIT group presented significantly elevated serum inflammatory cytokines including IL-1β, CXCL10, IL-7, IL-2, IL-10, IL-17, and TNF-α, and upregulation of TNF-α, IFN-γ, CXCR4, and IL-17 in AIT thyroid tissues (Fig. 2A and B). Descriptive data of cytokine values are presented in Supplementary Table 5. After adjusted for age, sex, BMI, and the time of sample collection, the expression of BMAL1 was negatively correlated with the levels of TPOAb (β = −0.526, P < 0.001) and IL-2 (β = −0.417, P = 0.004); the expression of PER2 was negatively associated with serum levels of TPOAb (β = −0.335, P = 0.032) and CXCL10 (β = −0.316, P = 0.044); and the mRNA expression of CXCR4 (β = −0.38, P = 0.009) in thyroid tissue (Fig. 2C). No significant association was found between other clock genes and inflammatory parameters (data not shown).
Association between inflammatory biomarker and mRNA levels of clock genes. (A) Elevated serum inflammatory cytokines in AIT patients compared with control group. (B) Increased mRNA expression of pro-inflammatory genes in thyroid samples from AIT patients compared with control group. n = 25–30/group (C) Associations of BMAL1 and PER2 with inflammatory biomarkers, multivariate linear regression models were adjusted for age, sex, BMI, and time of sample collection. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; two-way significance was calculated by Mann–Whitney U test.
Citation: European Thyroid Journal 12, 5; 10.1530/ETJ-23-0035
Diurnal inflammatory rhythmicity and effects of light disruption in EAT mice
To explore the diurnal rhythms in AIT, we employed the EAT mouse model. We first investigate the diurnal variations in the severity of EAT. A mixture of adjuvant and pTg administration led to a significant elevation of thyroid autoantibodies and destruction of thyroid follicular architecture. Neither the NC nor the EAT group exhibited significant rhythmicity in TgAb levels (Cosinor P > 0.05). While TgAb levels in the EAT group were slightly higher at ZT0 compared to ZT12, there were no significant differences observed between the two groups (Fig. 3A and B). Analysis of serum circulating cytokines showed that within the NC group, the levels of TNF-α were higher at ZT6, while IL-17, IL-6, and TGF-β were significantly higher at ZT18. Compared with the NC group, the levels of IL-17, IL-6, TGF-β, IFN-γ, and TNF-α significantly were elevated in EAT mice. In addition, the EAT group showed significantly higher IL-10 and TNF-α at ZT6 and higher IFN-γ at ZT18 (Fig. 3C).
Diurnal inflammatory responses in EAT. (A) Histopathology for thyroid tissues from NC and EAT mice at four Zeitgeber time points (ZT) (hematoxylin–eosin staining, ×400; scale bar = 50 μm). (B) Serum levels of TgAb in NC and EAT mice at four time points. (C) Comparison of serum levels of TgAb and inflammatory cytokines in EAT and NC mice at ZT6 and ZT18. Statistical significance was determined using Mann–Whitney U test (n = 6/group), and the results were represented as * P < 0.05, **P < 0.01, or ns (no significant difference); #P < 0.0, ##P < 0.01, and ###P < 0.001 indicate significant differences between NC and EAT group at identical time points, n = 5–6/group. EAT, experimental autoimmune thyroiditis; NC, normal control.
Citation: European Thyroid Journal 12, 5; 10.1530/ETJ-23-0035
Given the bidirectional relationship between circadian clock and inflammation, we subsequently examined the effects of LS (simulating night-shift conditions) on thyroid inflammation. In the control group, LS did not result in significant changes in thyroid morphology or levels of TgAb (Fig. 4A, B, and C). However, EAT mice kept in LS environment showed more serious architecture destruction and diffuse lymphocytic infiltration compared with normal light–darkness EAT mice (Fig. 4A and B). In line with this result, the levels of TgAb significantly increased in EAT mice exposure to LS, indicating enhanced thyroid autoimmune response under LS condition (Fig. 4C). The levels of serum cortisol, a biomarker reflecting both central clock and stress, were increased in LS group compared with NC mice. However, cortisol levels were comparable between EAT mice housed under normal light–darkness and LS, indicating stress related to thyroid inflammation overweight the influence of light disruption (Fig. 4D). Analysis of serum cytokines showed that LS resulted in a slight elevation of TNF-α (both in EAT and control mice) and IFN-γ (only in control group) (Fig. 4E).
Exposure to light shift aggravates thyroid inflammation in EAT mice. (A) Histopathology for thyroid tissues from control group (NC), mice under light shift (LS), mice with EAT under normal light (EAT) and mice with EAT under light shift (LS + EAT) (hematoxylin–eosin staining, ×400; scale bar = 50 μm; yellow arrow indicated inflammatory thyroid tissue with absent thyroid follicular architecture and lymphocytic infiltration). (B) EAT score for mice in NC, LS, EAT, and EAT + LS group. (C) Concentrations of serum TgAb in NC, LS, EAT, and EAT + LS group determined by ELISA. (D) Concentrations of serum cortisol in NC, LS, EAT, and EAT + LS group determined by ELISA. (E) Levels of inflammatory cytokines in NC, LS, EAT, and EAT + LS group determined by ELISA. (*on top of each bar indicated significant differences compared with NC group). n = 4–6/group; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; two-way significance was calculated by Mann–Whitney U test. EAT, experimental autoimmune thyroiditis; LS is the exposure to 4 weeks of light shift; NC, normal control (all samples were taken at ZT0).
Citation: European Thyroid Journal 12, 5; 10.1530/ETJ-23-0035
Disruption of thyroid clock by EAT and light shift
Alterations in the severity of EAT induced by LS further prompted us to analyze the change of thyroid clock genes in mice exposed to LS. Consistent with previous data, cosinor analysis and one-way ANOVA analysis confirmed rhythmicity of Bmal1, Clock, Per2, Cry1, Rev-erb,and Ror expression over the 24-h period in thyroid of NC mice (Fig. 5, Supplementary Fig. 4). As shown in Supplementary Table 6, pTg injections led to absent oscillations of Bmal1 expression, while LS conditions led to the loss of rhythmicity in Per2 and Cry1 expression in LS–NC mice. Through circadian analysis of each gene, we observed EAT and LS individually or in combination had distinct effects on the acrophase (peak), amplitude, and rhythmicity of different clock genes.
Effects of EAT and light shift on murine thyroid clock gene expression. n = 5–6/group. EAT, experimental autoimmune thyroiditis; LS is the exposure to 4 weeks of light shift; NC, normal control .
Citation: European Thyroid Journal 12, 5; 10.1530/ETJ-23-0035
By two-way ANOVA analysis, the expression of Clock, Per2, Cry1, Rev-erb, and Ror showed significant variations with LS, and LS had significant interaction with time for all six clock genes analyzed (P < 0.05). The expression of Clock, Rev-erb, and Ror genes showed significant variations with EAT, and EAT had significant interaction with time for Bmal1, Clock, Cry1, and Ror (P < 0.05). In summary, both light disruption and pTg injections had significant effects on the expression of circadian clock genes in mouse thyroid. Additionally, the interaction between these treatments and time further modulated the expression patterns of clock genes (Supplementary Table 7).
Discussion
In the current study, we demonstrated a broad disruption of clock gene profiles within thyroid samples from AIT patients. Temporal analyses of clock gene transcripts demonstrated the robust daily oscillations of clock genes expression within normal murine thyroid tissue, which was disturbed in the EAT mouse model. Although circadian disruption did not directly induce the development of thyroid autoimmunity, EAT mice housed under LS showed more severe thyroid lymphocytic infiltration, accompanied by overproduction of TgAb and cytokines. These results demonstrated the reciprocal connection between circadian disruption and autoimmune thyroid disease.
Since the discovery of circadian clockwork in Drosophila (23), a growing body of evidence described the circadian expression of clock genes and clock-controlled genes in peripheral tissues (6). Fahrenkrug and colleagues first demonstrated the 24-h antiphase oscillations of Bmal1 and Per1 in rat thyroid and revealed that the thyroid clock was sustained even after abolishing the regulation of SCN by hypophysectomy (24). The existence of thyroid clock was further supported by the study from Georg and colleagues which showed identical rhythms of Per1/2 and Bmal1 expression in thyroid between wild-type and VPAC2-receptor (loss of SCN clock)-deficient mice (14). Consistent with previous findings, our study revealed a clear time-dependent oscillation of clock genes, with a repressed expression of Bmal1 and Clock at darkness phase.
Besides temporal analyses of clock transcripts in thyroid, we also extend this observation to the circadian rhythms in thyroid autoimmunity. Studies on patients and mouse models consistently reported that acute or chronic inflammation in RA, osteoarthritis, inflammatory bowel disease, and atherosclerosis can directly dampen the peripheral clock network (25, 26, 27, 28, 29). Lipopolysaccharide challenge can lead to a 6-h retrograde shift in Per1/2 mRNA expression and overall reduced expression of Bmal1 and Per1/2 in the spleen (30). In agreement with previous findings on the anti-inflammatory effects of most core clock genes, our study demonstrated that the expression of Bmal1 and Per2 was significantly reduced in AIT patients and were associated with serum TPOAb levels. Our studies in mouse models consistently showed a broad disruption of clock genes within inflamed thyroid tissue. Taking together these findings, we postulate that the expression of clock genes may exhibit a potential regulatory effect in thyroid autoimmunity.
As an internal timekeeper of physiology, the circadian system also in turn modulates the immune system by clock-controlled innate immune cell movement, pathogen responses, and lymphocyte trafficking (11). Inflammation drives a vicious cycle of immune response and disrupted circadian clock rhythm (31, 32, 33, 34). Animal and human studies have shown that frequent night shifts, air travel, and sleep disorders were associated with an increased incidence of a broad variety of chronic inflammatory diseases (17). In terms of thyroid disease, night shift work has been associated with increased risk of elevated TSH levels and malignant thyroid nodules (35, 36, 37, 38). The present study is the first to investigate the potential effect of LS on EAT in mice. Significantly, we found that the time-of-day rhythmicity in thyroid clock was lost in animals housed under LS environment. The exacerbation of EAT by LS replicates the clinical evidence on the association between night shift and prevalence of thyroid disease and highlights the complex nature of the interactions between the molecular clock and inflammatory reactions (39, 40). These findings suggest that both external environmental factors (such as light disruption) and immune-related interventions (such as antigen injection) can impact the circadian rhythm of clock genes in the thyroid of mice.
However, our study has several limitations. First, the individual's chronotype is known to influence the expression of many genes and levels of melatonin. Since we did not measure the patients' chronotype and were unable to control the time of sample collection, we cannot fully account for the potential influence of the timing on our results. As most of our human samples were collected during the daytime, our analysis may be insufficient to detect a meaningful association between these parameters and time. Additionally, although we employed a multiple linear regression analysis incorporating the time of sample collection, BMI, age, and sex as covariates, the interpretation of our correlation results remains complex due to the intricate nature of circadian rhythms and the possible confounding factors. Secondly, while our study presented the diurnal variations of biological parameters, a 6-h time interval has a very low power to detect rhythmicity. As the samples were collected from an animal model, it was imperative to obtain as much data as possible within a limited timeframe. Thirdly, instead of using the thyroid nodule tissue itself, we chose nodule-adjacent tissue as control samples because the nodule tissue is usually preserved for pathological examinations. However, it is important to note that the presence of thyroid nodules can affect the surrounding microenvironment, resulting in alterations in gene expression, immune responses, and cellular functions (41). This intermediate phenotype in tumor adjacent tissues may add complexity to the interpretation of our findings. Fourthly, in light of the increased prevalence of AIT in females (1) and to minimize the influence of sex hormones, we used female mice in our study referring to previous EAT models (42). While this choice allows us to focus on AIT in a population more relevant to the human context, it is important to acknowledge that our findings may have limited applicability to male populations.
In conclusion, this study suggests that thyroid-intrinsic clock was dampened in AIT patients and mouse models. Furthermore, we identified LS as a risk factor for the exacerbation of AIT. Our findings indicate the potential role of circadian disruption in the progression of AIT, providing opportunities to alleviate autoimmune thyroid disease from the view of chrono-medicine.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ETJ-23-0035.
Declaration of interest
The authors declare that they have no competing interests.
Funding
This study was funded by the National Natural Science Foundation of China (No. 81870538) and GDPH Supporting Fund for Talent Program (No.KJ012020629).
Author contribution statement
HG contributed to the conception and design of the study. LH, HL, and QL collected the tissue samples. JF and ZF performed the experiments and wrote the original draft. HG and YL helped edit and revise the manuscript. All authors have read and approved the final manuscript.
Acknowledgements
We are grateful to the participants in this study.
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