Abstract
Graves’ disease (GD) is characterized by dysregulation of the immune system with aberrant immune cell function. However, there have been few previous studies on the role of monocytes in the pathology of GD. The object of this study was to investigate whether and how monocytes participate in GD pathology. CD14+ monocytes were isolated from untreated initial GD patients and healthy controls. Then, RNA-seq was performed to investigate changes in global mRNA expression in monocytes and found that type I interferon (IFN) signalling was among the top upregulated signalling pathways in GD monocytes. Type I IFN-induced sialic acid-binding immunoglobulin-like lectin1 (SIGLEC1) expression was significantly upregulated in untreated GD patients and correlated with thyroid parameters. Patient serum SIGLEC1 concentrations were reduced after anti-thyroid drug treatment. Inhibiting SIGLEC1 expression could inhibit proinflammatory cytokine (IL-1β, IL-6, IL-8, IL-10 and M-CSF) expression in monocytes. In conclusion, our study suggested that type I IFN-mediated monocyte activation could have a deleterious effect on the pathogenesis of GD. These observations indicated that the inhibition of type I IFN-activated monocytes/macrophages could have a therapeutic effect on GD remission.
Introduction
Graves’ disease (GD) is a common autoimmune disorder that affects ~2% of women and 0.2% of men globally (Smith & Hegedüs 2016, Taylor et al. 2018, Davies et al. 2020). Although the aetiology of GD remains unknown, strong evidence indicates that it is caused by genetic components combined with environmental factors in immunologically susceptible individuals (Davies et al. 2020). GD is characterized by immune cell infiltration in the thyroid gland and the production of thyroid-stimulating hormone receptor antibodies (TRAbs). Over time, emerging evidence has shown an association between viral infection and autoimmune thyroid disease (AITD) (Yin et al. 2014, Caselli et al. 2017, Hammerstad et al. 2017, 2020).
Interferon (IFN), a stress-related cytokine secreted during viral infections, has been suspected to be associated with the pathogenesis of multiple autoimmune diseases and has been associated with the onset and development of specific autoimmune diseases, including AITDs, but the role of IFN and the contribution of specific cell types have not been fully clarified (Banchereau & Pascual 2006, Kuang et al. 2010, Mao et al. 2011, Rönnblom & Eloranta 2013, Faustino et al. 2018). The IFN family includes type I, type II and type III IFN subgroups. Our previous work showed that increased plasmacytoid dendritic cells (pDCs) were one source of the increased serum IFN-α in untreated initial GD (uGD) patients, which then caused the upregulation of IFNα-inducible genes (IFIGs) in peripheral blood mononuclear cells (PBMCs) from uGD patients (Kuang et al. 2010, Mao et al. 2011). In uGD patients, increased pDCs and IFN-α secretion impaired the inhibitory activity of CD4+CD25+ Treg cells and promoted T cell proliferation (Mao et al. 2011). In addition, IFN-α can also upregulate MHC-II antigens and enhance autoantigen presentation of thyroid-stimulating hormone receptor (TSHR) on thyrocytes (Kuang et al. 2010). Furthermore, others have shown that IFN-α can induce the lysosomal degradation of thyroglobulin to release pathogenetic peptides that can trigger thyroid autoimmunity (Faustino et al. 2018). Monocytes, which are a special kind of antigen-presenting cell and can present antigens to activate CD4+ T cells upon stimulation with IFNα (Blanco et al. 2001), have an unknown role in IFN-mediated GD pathology. Thus, we sought to identify monocyte-specific IFN signatures in the pathology of GD.
In the present study, we performed global RNA sequencing of CD14+ monocytes from uGD patients and elucidated how monocytes were associated with GD pathology. We found increased percentages of CD14+ monocytes and upregulated monocyte type I IFN signalling in uGD patients. SIGLEC1, a CD14+ monocyte/macrophage-restricted receptor and an interferon-stimulated gene (ISG) that is upregulated by type I IFN, was increased in uGD patients at both the monocyte mRNA and serum levels. Inhibiting monocyte SIGLEC1 expression caused decreased production of proinflammatory cytokines by monocytes, which could be a potential therapeutic strategy for GD.
Materials and methods
Human subjects
In this study, 47 uGD patients and 18 age- and sex-matched healthy controls (HCs) were enrolled. GD diagnosis was based on typical clinical manifestations and laboratory examinations. Laboratory examinations included increased serum concentrations of free triiodothyronine (FT3) and free thyroxine (FT4), decreased serum concentrations of sensitive thyrotropin (sTSH) and high titres of TRAbs. Clinical manifestations included increased appetite, increased sweating, weight loss, heat intolerance, fatigue, muscle weakness, tremors and a diffusely enlarged thyroid gland. Patients with uGD were all newly diagnosed and had not been treated with the anti-thyroid drug (ATD) methimazole (MMI) in our study. Patients were considered to be in remission (nGD, TRAb-negative GD) when their thyroid function returned to normal (sTSH, FT3 and FT4 within the reference range and negative for TRAbs) after routine ATD treatment. Furthermore, a group of subjects without thyroid disease were enrolled as HCs. These subjects had no personal or family history of thyroid disease, had serum TSH and FT3/FT4 levels within the reference ranges, were negative for thyroid antibodies (TRAbs), thyroid peroxidase antibodies (TPOAbs) and thyroglobulin antibodies (TGAbs)) and had normal thyroid ultrasound imaging. Informed written consents were obtained from all participant in accordance with the Declaration of Helsinki. The study was approved by the Research Ethics Board of Ruijin Hospital.
Peripheral blood mononuclear cell and CD14+ monocyte isolation
Briefly, fresh PBMCs from GD patients and HCs were isolated by gradient centrifugation on Ficoll-Paque Plus (GE Healthcare). To purify CD14+ cells from fresh PBMCs, CD14+ monocytes were isolated by positive selection magnetic sorting using human CD14 microbeads and LS columns (Miltenyi Biotec, Cologne, Germany) according to the manufacturer’s instructions. The purity of CD14+ monocytes was greater than 95%, as examined by flow cytometry.
Human serum SIGLEC1 analysis
Quantitative measurement of serum SIGLEC1 (CD169) levels was performed using a human CD169 ELISA kit (SIGLEC1) (Abcam) according to the manufacturer’s instructions. For each participant, 100 µL of serum was used.
CD14+ monocyte culture and treatments
Fresh isolated CD14+ monocytes from HCs and uGD patients were cultured in 12-well plates in culture medium (RPMI 1640, 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin) at 37°C in a humidified 5% CO2 atmosphere. After being incubated overnight, isolated CD14+ monocytes were treated with anti-IgG antibodies or anti-Siglec-1 antibodies (2 μg/mL, NB600-534; Novus Biologicals, Centennial, CO, USA) for 72 h. After treatment, CD14+ monocytes were collected for further gene expression analysis.
Monocyte RNA sequencing analysis
Freshly prepared CD14+ monocyte samples from uGD patients and HCs (n = 3) were used for RNA extraction. Then, RNA sequencing (RNA-seq) analysis was performed by Cloud-seq Biotech Inc. (Shanghai, China) according to published procedures. rRNA was removed from the total RNA with an NEBNext rRNA depletion kit according to the manufacturer’s instructions. RNA libraries were constructed, and the libraries were controlled for quality and quantified. Library sequencing was performed on an Illumina HiSeq instrument with 150 bp paired end reads. Then, guided by the Ensembl GTF gene annotation file, Cuffdiff software (part of Cufflinks) was used to obtain the gene-level fragments per kilobase of exon model per million mapped reads (FPKM) as the mRNA expression profiles. Fold changes and P values were calculated based on the FPKM. The mRNA expression profiles were compared between the uGD and HC groups.
Quantitative real-time PCR analysis
Total monocyte RNA was extracted using TRIzol reagent (Invitrogen). RT and quantitative real-time PCR were performed as previously described (Qi et al. 2017). PCR was performed in duplicate, and the results were calculated by determining the ΔΔCt value. The expression levels were normalized to individual GAPDH or actin levels. The primers used in this study are listed in Supplementary Table 1 (see section on supplementary materials given at the end of this article).
Statistical analysis
All statistical analyses were performed using SPSS software or GraphPad Prism 8. The data are presented as the means ± s.e.m. if normally distributed and as medians and interquartile ranges (median, 25th percentile to 75th percentile) if not normally distributed. Pearson’s chi-square test was used to determine the statistical significance of differences among proportions. Associations between clinical features and the results were examined using binary logistic regression. An unpaired Student’s t-test or the Mann–Whitney U-test was performed to compare differences between two groups. Multivariate analysis was performed by multiple linear regression analysis. In the area under the ROC curve (AUC) analysis, sensitivity and specificity were evaluated. A value of P < 0.05 was considered statistically significant.
Results
RNA-seq analysis identified activated type I IFN signalling pathway in monocytes from uGD patients
To determine the global gene expression profile in uGD monocytes, we isolated CD14+ monocytes from freshly isolated PBMCs from uGD patients and HCs (n = 3) to perform RNA sequencing analysis.
We identified a total of 888 differentially expressed genes (DEGs) (fold change > 2, P < 0.05) in monocytes from uGD patients and HCs. Among them, 704 (79.3%) were downregulated and 184 (20.7%) were upregulated (Fig. 1A). Then, we performed Gene Ontology (GO) analysis of these DEGs (Fig. 1B). Signalling pathways associated with the type I IFN signalling pathway, the defence response to viruses, the response to external stimuli, the innate immune response and the cellular response to cytokine stimuli were significantly upregulated (Fig. 1B). Among them, the most highly upregulated biological process was the type I IFN signalling pathway (Fig. 1B). Pathways such as leukocyte activation regulation, T cell receptor signalling and antigen receptor-mediated signalling were significantly downregulated (Fig. 1B).
RNA-seq analysis of monocytes identified upregulated type I IFN signalling in uGD. (A) Scatter plots of the relative expression levels of 888 differentially expressed genes identified by RNA-seq analysis of healthy control (HC) and uGD monocytes (n = 3). Upregulated genes are marked in red (184 genes); downregulated genes are marked in blue (704 genes) (fold change > 2, P < 0.05). (B) GO analysis of differentially expressed genes identified by RNA-seq. (C) Heatmap showing the relative expression levels of genes in the type I IFN signalling pathway between HC and uGD patients (n = 3). A full colour version of this figure is available at https://doi.org/10.1530/JOE-21-0453.
Citation: Journal of Endocrinology 255, 1; 10.1530/JOE-21-0453
Our previous work had already reported the upregulation of IFN-α and IFIGs (i.e. IFIT1 and IFIT4) in PBMCs from uGD patients (Mao et al. 2011). However, which immune cells dominate IFN signalling-induced pathology remains unclear. As shown in Fig. 1C, we found that genes associated with type I IFN signalling activation (STAT1, STAT2, IRF7, IFIT1, IFIT2 and IFIT3) were significantly increased in uGD monocytes. Interestingly, SIGLEC1, a CD14+ monocyte/macrophage-restricted receptor gene associated with monocyte/macrophage activation (Xiong et al. 2014), was one of the top IFN-regulated genes identified (Fig. 1C).
Increased SIGLEC1 expression in uGD monocytes and infiltration of SIGLEC1+ cells in GD thyroid tissues
A previous study reported the predictive role of elevated SIGLEC1 mRNA in PBMCs in GD relapse (Hashimoto et al. 2018). However, the role of SIGLEC1 in GD onset remains unknown. Thus, we enrolled a total of 47 uGD patients and 18 HCs in this study to validate our sequencing results. General patient characteristics were shown in Table 1. The two groups were age- and sex-matched. Thyroid parameters (FT3 and FT4) and thyroid autoantibodies (TRAbs, TGAbs and TPOAbs) were significantly increased, while sTSH levels were significantly decreased in uGD patients compared to HCs (Table 1). Moreover, we observed an increased percentage of peripheral monocytes in our population (6.26 ± 1.32% vs 11.43 ± 3.20% in uGD patients, P < 0.01, Supplementary Fig. 1A), while the absolute number of monocytes did not significantly increase (0.39 ± 0.11 × 109/L vs 0.91 ± 1.59 × 109/L in uGD patients, P = 0.16, Supplementary Fig. 1B).
Clinical characteristics of the untreated Graves’ disease patients and healthy controls.
| Healthy controls | Untreated-GD | P value | |
|---|---|---|---|
| n (famale/male) | 18 (14/4) | 47 (44/3) | 0.124 |
| Age (years) | 36.81 ± 17.78 | 38.94 ± 12.92 | 0.580 |
| FT3 (pmol/L) | 4.13 ± 0.40 | 27.49 ± 13.54 | P < 0.001a |
| FT4 (pmol/L) | 12.81 ± 1.09 | 44.57 ± 10.92 | P < 0.001a |
| TSH (mIU/L) | 1.91 ± 0.89 | 0.0009 ± 0.0014 | P < 0.001a |
| TRAb (IU/L) | 0.31 ± 0.05 | 16.44 ± 9.20 | P < 0.001a |
| TPOAb (IU/mL) | 1.28 ± 2.32 | 238.61 ± 269.47 | P < 0.001a |
| TGAb (IU/mL) | 3.57 ± 4.25 | 140.82 ± 184.39 | P < 0.01b |
aP < 0.001; bP < 0.01; P values were calculated from ANOVA (between categories), after adjustment for age and gender.
IFNs induce the expression of genes through the type I IFN receptor (IFNR1). By using RT-PCR, we found that IFN-alpha/beta receptor 1 (IFNAR1)and SIGLEC1 mRNA levels were significantly increased in uGD monocytes (Fig. 2A and B) (n = 18-47). We also checked the expression of other IFN-related genes in our GD monocyte samples. We checked the expression of those differentially expressed genes (STAT1, STAT2, IFI6, IFI35, IFIT1, IFIT2, IFIT3 and IFITM3) identified from our RNA-seq results. We found the significant upregulation of IFI35, IFITM3, IFI6 and STAT2 mRNA expression in untreated GD samples (Supplementary Fig. 2).
Increased IFIG expression in monocytes from uGD patients. (A and B) RT-PCR analysis of monocytes from HC and uGD patients showing increased IFNAR1 (A) and Siglec-1 (B) mRNA expression in uGD monocytes. (n = 18-47). (C) Increased serum SIGLEC-1 expression in HC (n = 18) and uGD (n = 47) patients. (D and E) ROC curves of monocyte SIGLEC1 mRNA (D) and serum SIGLEC1 levels (E) as a screening tool for uGD patients. (F) Serum SIGLEC1 levels in GD patients before and after ATD treatment. uGD, untreated GD; nGD, TRAb-negative GD. (G) Immunofluorescent staining of SIGLEC1 in thyroid tissues from patients with GD and HCs. The data are presented as the mean ± s.e.m. of independent experiments as indicated, ** P < 0.01. Student’s t-test. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. A full colour version of this figure is available at https://doi.org/10.1530/JOE-21-0453.
Citation: Journal of Endocrinology 255, 1; 10.1530/JOE-21-0453
Moreover, serum SIGLEC1 levels were obviously increased in uGD patients (446.43 ± 301.88 vs 2668.25 ± 2762.02 pg/mL in uGD patients, P < 0.01, Fig. 2C). We further performed immunostaining for the monocyte/macrophage marker F4/80 in GD thyroid tissue sections and observed increased macrophage infiltration in GD thyroid tissue (Supplementary Fig. 3). Moreover, we found that the number of SIGLEC-1+ CD14+ cells was significantly increased in GD thyroid sections (Fig. 2G), whereas these SIGLEC-1+ macrophages could barely be found in normal thyroid tissues. These data indicated activated type I IFN signalling and increased SIGLEC1 expression on monocytes from uGD patients.
Correlation between SIGLEC1 and thyroid parameters in uGD patients
To investigate the correlation between SIGLEC1 expression and thyroid parameters, we performed linear regression analyses (Fig. 3A, B, C, D, E, F, G and H). FT4 was found to be positively correlated with monocyte SIGLEC1 mRNA levels (R2 = 0.185, P < 0.01), while TSH was negatively correlated with monocyte SIGLEC1 mRNA levels in uGD patients (R2 = 0.130, P < 0.05) (Fig. 3B and C). No correlation between monocyte SIGLEC1 mRNA levels and patient FT3 or TRAb levels was found (Fig. 3A and D). Moreover, serum SIGLEC1 levels were positively correlated with serum FT4 and TRAb levels in uGD patients (R2 = 0.075, P < 0.05 for FT4; R2= 0.383, P < 0.01 for TRAbs) (Fig. 3F and H). No linear correlation between serum SIGLEC1 levels and FT3 or TSH was found (Fig. 3E and G).
Correlation between SIGLEC1 mRNA, serum SIGLEC1 levels and thyroid parameters in uGD patients. (A, B, C and D). Correlation between FT3, FT4, sTSH and TRAb levels and SIGLEC1 mRNA expression in monocytes from uGD patients. (n = 47). (E, F, G and H) Correlation between FT3, FT4, sTSH and TRAb levels and serum SIGLEC1 concentrations in uGD patients. (n = 47 for each plot). *P < 0.05, **P < 0.01.
Citation: Journal of Endocrinology 255, 1; 10.1530/JOE-21-0453
The positive association between TRAbs and SIGLEC1 levels led us to generate ROC plots to evaluate the diagnostic performance of SIGLEC1 in GD initiation. The AUC of monocyte SIGLEC1 mRNA levels and serum SIGLEC1 levels was 0.965 and 0.935, respectively (Fig. 2D and E). Moreover, we followed a group of patients until their thyroid function returned to normal ranges and their TRAb antibody turned to negative (nGD) after routine ATD treatment. We found reduced serum SIGLEC1 level after ATD treatment (Fig. 2F).
Inhibiting SIGLEC1 expression reduced cytokine production in monocytes
Monocyte activation manifests as increased proinflammatory cytokine and chemokine production that can therefore lead to the initiation of the innate immune response (Gaidt et al. 2016). We examined whether inhibiting monocyte SIGLEC-1 could ameliorate the inflammatory response of GD monocytes.
The monocyte proinflammatory cytokines M-CSF, IL-1β, IL-6, IL-8, IL-10 and TNF-α were measured. M-CSF, IL-1β, IL-6. IL-8 and IL-10 mRNA expression was significantly increased in uGD monocytes compared to HCs (Fig. 4A, B, C, D and E). Then, to functionally block the expression of SIGLEC1, isolated monocytes were treated with a blocking antibody against SIGLEC1 (2 µg/mL) for 72 h. Significantly reduced M-CSF, IL-1β, IL-6. IL-8 and IL-10 mRNA expression after SIGLEC1 inhibition was observed in monocytes from uGD patients (Fig. 4A, B, C, D and E). However, we did not observe significant changes in TNF-α after monocyte SIGLEC1 blockade (Fig. 4F).
Inhibition of SIGLEC1 reduced monocyte proinflammatory cytokine production. (A, B, C, D, E and F) Monocytes were isolated from HCs and uGD patients and treated with anti-SIGLEC1 or anti-IgG antibodies for 72 h. RT-PCR showing the relative mRNA expression of monocyte proinflammatory cytokines (M-CSF, IL-1β, IL-6, IL-8, IL-10 and TNF-α) after SIGLEC1 inhibition. (n = 3). The data are presented as individual values and mean ± s.e.m. of independent experiments as indicated, ** P < 0.01. A full colour version of this figure is available at https://doi.org/10.1530/JOE-21-0453.
Citation: Journal of Endocrinology 255, 1; 10.1530/JOE-21-0453
Discussion
In this study, we performed global RNA sequencing of CD14+ monocytes from uGD patients and found upregulated monocyte type I IFN signalling in uGD. We found that the CD14+ monocyte/macrophage-restricted receptor SIGLEC1, which was upregulated by type I IFN, was increased in uGD patients. Moreover, the upregulation of proinflammatory cytokines in uGD monocytes could be reversed by inhibiting monocyte SIGLEC-1. This finding suggested the potential therapeutic effect of blocking monocyte IFN/SIGLEC1 signalling in GD treatment.
Many studies have reported the pathogenic role of CD4+ T cell and B cell dysfunction in GD. In addition to T and B cells, monocytes are also key components of the innate immune system and are essential for the recognition of self-antigens and the regulation of the adaptive immune response (Karlmark et al. 2012, Gaidt et al. 2016). Undifferentiated peripheral monocytes are heterogeneous and can be classified into three main subsets: classical (CD14++CD16−) monocytes, intermediate (CD14++CD16+) monocytes and non-classical (CD14+CD16+) monocytes (Ziegler-Heitbrock et al. 2010, Ziegler-Heitbrock & Hofer 2013). Peripheral monocytes give rise to macrophages and DCs. After presenting antigens, monocytes are the major sources of inflammatory cytokines and are responsible for T or B cell activation. Peripheral monocytes have been shown to participate in immune abnormalities in various autoimmune diseases (Hohl et al. 2009, Karlmark et al. 2012, Rossol et al. 2012, Chuluundorj et al. 2014, Cox et al. 2015). During the onset of infection with various pathogens or in response to inflammation, monocytes initiate effector functions and exert proinflammatory effect by producing proinflammatory cytokines such as IL-6 and TNF-α. Hohl et al. reported that monocytes are essential for the priming and expansion of CD4+ T cell responses during respiratory fungal infection (Hohl et al. 2009). In IgA nephropathy patients, expansion of the CD14+CD16+ monocyte subset resulted in an increased apoptotic phenotype in these cells and thus contributed to disease pathogenesis (Cox et al. 2015). However, the role of monocytes/macrophages in GD has not been fully elucidated. Our previous work has already reported different subsets of CD14+ monocytes in the peripheral blood of uGD patients. We showed that CD14++ CD16+ monocytes (intermediate monocytes) were increased and CD14++ CD16- monocytes (classical monocytes) were decreased in untreated GD (both P < 0.001) (Chen et al. 2021), suggesting the participation of intermediate monocytes in GD immune responses.
In this study, the transcriptome profile of monocytes revealed the activation of type I IFN signalling and the deleterious effect of SIGLEC1 upregulation. IFN-α and type I IFN signalling have been proven to be involved in GD in different ways. In thyroid tissues, IFN-α can induce thyrocytes to express MHC-II antigens and TSHR to become antigen-presenting cells (Kuang et al. 2010). In the periphery, IFN-α can repress the inhibitory activity of Treg cells, which then contribute to the loss of peripheral self-tolerance (Mao et al. 2011). Here, we showed hyperactivity of type I IFN signalling in monocytes. The viral immune response proteins signal transducer and activator of transcriptions 1 and 2 (STAT1 and STAT2), which are downstream of IFN-I, were induced in uGD monocytes. Upon activation by IFNs, the cytoplasmic protein STAT1 translocates to the nucleus to initiate antiviral responses. Recently, Weider et al. also showed STAT1 positivity in thyrocytes in uGD, suggesting an active IFN-driven immune response in thyroid tissues (Weider et al. 2021). Our results also supported the hypothesis that viral infection was involved in the onset of GD and indicated that this effect might be caused at least partially by monocyte-mediated immunopathological processes.
SIGLEC1, which is also known as sialoadhesin or CD169, is a CD14+ monocyte/macrophage-restricted receptor and can bind to granulocytes, erythrocytes, B cells and CD43 on T cells (O’Neill et al. 2013). SIGLEC1 is mainly expressed on tissue resident macrophages, but it can also be expressed on circulating monocytes under inflammatory conditions. Increased SIGLEC1 expression in human monocytes is predominantly induced by type 1 IFN (Xiong et al. 2009, O’Neill et al. 2013). SIGLEC1+ cells have dual physiological functions, acting as innate ‘flypaper’ by preventing the systemic spread of lymph-borne pathogens and as critical gatekeepers at the lymph-tissue interface to facilitate the recognition of particulate antigens by B cells and initiate humoural immune responses. Previous studies have shown that in addition to viral infection, SIGLEC1 plays a detrimental role in multiple inflammatory and autoimmune diseases, including rheumatoid arthritis (Xiong et al. 2014), systemic sclerosis (SSc) (York et al. 2007) and primary biliary cirrhosis (Bao et al. 2010). Recently, Hashimoto et al. reported the role of SIGLEC1 in predicting GD relapse risk (Hashimoto et al. 2018). However, whether and how SIGLEC1 is involved in the initiation of GD remains unknown. In this study, we identified increased monocyte SIGLEC1 mRNA expression and soluble serum SIGLEC1 in newly diagnosed GD. The increase in soluble serum SIGLEC1 levels was strongly correlated with serum TRAb levels in uGD patients. We also found decreased serum SIGLEC1 expression after ATD treatment. In vitro, we observed that inhibiting monocyte SIGLEC1 expression with a blocking antibody decreased monocyte proinflammatory cytokine levels. Proinflammatory cytokines participate in different stages of immune responses and in the pathogenesis of AITD. For example, IL-1β, a potent proinflammatory cytokine produced by activated monocytes/macrophages, is an important mediator of the inflammatory response and can subsequently activate T cells and B cells (Masters et al. 2009, Estruch et al. 2015, Gaidt et al. 2016). Over time, the conventional treatments of GD, including ATDs, radioiodine or surgery, have not changed. For many patients, treatment is either ineffective or leads to lifelong thyroid hormone replacement therapy in the case of the latter two options (Ferrari et al. 2020). Understanding the immunobiology of GD and identifying novel therapeutic strategies would benefit the treatment of many patients. Drugs targeting major players in GD aetiology, including monocytes/macrophages, T cells and B cells, such as etanercept, adalimumab (anti-TNFα), tocilizumab (anti-IL-6) and iscalimab (anti-CD40), are being assessed for the treatment of GD and Graves’ orbitopathy (Davies et al. 2020, Ferrari et al. 2020). These therapies provide new opportunities to replace the current inadequate treatment for some Graves' patients, but the efficacy and safety of these therapies still need to be verified. Our study revealed the hyperactivity of type I IFN signalling and a profound increase in SIGLEC1 mRNA expression in monocytes, and blocking SIGLEC1 decreased monocyte proinflammatory cytokine production. This finding suggested that SIGLEC1 might be a potential target for GD immunotherapy. However, further studies are needed to verify the clinical efficacy of blocking monocyte IFN/SIGLEC1 signalling in GD treatment.
In conclusion, our study increased our understanding of monocyte abnormalities in GD and identified the role of IFN/SIGLEC1 signalling in the pathology of GD, which might be a potential therapeutic target of GD.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-21-0453.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the National Natural Science Foundation of China Grants (81873637).
References
Banchereau J & Pascual V 2006 Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity 25 383–392. (https://doi.org/10.1016/j.immuni.2006.08.010)
Bao G, Han Z, Yan Z, Wang Q, Zhou Y, Yao D, Gu M, Chen B, Chen S, Deng A, et al.2010 Increased Siglec-1 expression in monocytes of patients with primary biliary cirrhosis. Immunological Investigations 39 645–660. (https://doi.org/10.3109/08820139.2010.485625)
Blanco P, Palucka AK, Gill M, Pascual V & Banchereau J 2001 Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus. Science 294 1540–1543. (https://doi.org/10.1126/science.1064890)
Caselli E, D'Accolti M, Soffritti I, Zatelli MC, Rossi R, Degli Uberti E & Di Luca D 2017 HHV-6A in vitro infection of thyrocytes and T cells alters the expression of miRNA associated to autoimmune thyroiditis. Virology Journal 14 3. (https://doi.org/10.1186/s12985-016-0672-6)
Chen X, Wang Y, Qi Y, Yan J, Huang F, Zhou M, Wang W, Ning G, Zhou Y & Wang S 2021 Expansion of inflammatory monocytes in periphery and infiltrated into thyroid tissue in Graves’ disease. Scientific Reports 11 13443. (https://doi.org/10.1038/s41598-021-92737-4)
Chuluundorj D, Harding SA, Abernethy D & La Flamme AC 2014 Expansion and preferential activation of the CD14(+)CD16(+) monocyte subset during multiple sclerosis. Immunology and Cell Biology 92 509–517. (https://doi.org/10.1038/icb.2014.15)
Cox SN, Serino G, Sallustio F, Blasi A, Rossini M, Pesce F & Schena FP 2015 Altered monocyte expression and expansion of non-classical monocyte subset in IgA nephropathy patients. Nephrology, Dialysis, Transplantation 30 1122–1232. (https://doi.org/10.1093/ndt/gfv017)
Davies TF, Andersen S, Latif R, Nagayama Y, Barbesino G, Brito M, Eckstein AK, Stagnaro-Green A & Kahaly GJ 2020 Graves’ disease. Nature Reviews: Disease Primers 6 52. (https://doi.org/10.1038/s41572-020-0184-y)
Estruch M, Rajamäki K, Sanchez-Quesada JL, Kovanen PT, Öörni K, Benitez S & Ordoñez-Llanos J 2015 Electronegative LDL induces priming and inflammasome activation leading to IL-1β release in human monocytes and macrophages. Biochimica et Biophysica Acta 1851 1442–1449. (https://doi.org/10.1016/j.bbalip.2015.08.009)
Faustino LC, Lombardi A, Madrigal-Matute J, Owen RP, Libutti SK & Tomer Y 2018 Interferon-α triggers autoimmune thyroid diseases via lysosomal-dependent degradation of thyroglobulin. Journal of Clinical Endocrinology and Metabolism 103 3678–3687. (https://doi.org/10.1210/jc.2018-00541)
Ferrari SM, Fallahi P, Elia G, Ragusa F, Camastra S, Paparo SR, Giusti C, Gonnella D, Ruffilli I, Shoenfeld Y, et al.2020 Novel therapies for thyroid autoimmune diseases: an update. Best Practice and Research: Clinical Endocrinology and Metabolism 34 101366. (https://doi.org/10.1016/j.beem.2019.101366)
Gaidt MM, Ebert TS, Chauhan D, Schmidt T, Schmid-Burgk JL, Rapino F, Robertson AA, Cooper MA, Graf T & Hornung V 2016 Human monocytes engage an alternative inflammasome pathway. Immunity 44 833–846. (https://doi.org/10.1016/j.immuni.2016.01.012)
Hammerstad SS, Stefan M, Blackard J, Owen RP, Lee HJ, Concepcion E, Yi Z, Zhang W & Tomer Y 2017 Hepatitis C virus E2 protein induces upregulation of IL-8 pathways and production of heat shock proteins in human thyroid cells. Journal of Clinical Endocrinology and Metabolism 102 689–697. (https://doi.org/10.1210/jc.2016-3403)
Hammerstad SS, Blackard JT, Lombardi A, Owen RP, Concepcion E, Yi Z, Zhang W & Tomer Y 2020 Hepatitis C virus infection of human thyrocytes: metabolic, hormonal, and immunological implications. Journal of Clinical Endocrinology and Metabolism 105 1157–1168. (https://doi.org/10.1210/clinem/dgz241)
Hashimoto K, Nishihara E, Matsumoto M, Matsumoto S, Nakajima Y, Tsujimoto K, Yamakage H, Satoh-Asahara N, Noh JY, Ito K, et al.2018 Sialic acid-binding immunoglobulin-like Lectin1 as a novel predictive biomarker for relapse in Graves’ disease: a multicenter study. Thyroid 28 50–59. (https://doi.org/10.1089/thy.2017.0244)
Hohl TM, Rivera A, Lipuma L, Gallegos A, Shi C, Mack M & Pamer EG 2009 Inflammatory monocytes facilitate adaptive CD4 T cell responses during respiratory fungal infection. Cell Host and Microbe 6 470–481. (https://doi.org/10.1016/j.chom.2009.10.007)
Karlmark KR, Tacke F & Dunay IR 2012 Monocytes in health and disease – minireview. European Journal of Microbiology and Immunology 2 97–102. (https://doi.org/10.1556/EuJMI.2.2012.2.1)
Kuang M, Wang S, Wu M, Ning G, Yao Z & Li L 2010 Expression of IFNalpha-inducible genes and modulation of HLA-DR and thyroid stimulating hormone receptors in Graves’ disease. Molecular and Cellular Endocrinology 319 23–29. (https://doi.org/10.1016/j.mce.2009.12.006)
Mao C, Wang S, Xiao Y, Xu J, Jiang Q, Jin M, Jiang X, Guo H, Ning G & Zhang Y 2011 Impairment of regulatory capacity of CD4+CD25+ regulatory T cells mediated by dendritic cell polarization and hyperthyroidism in Graves’ disease. Journal of Immunology 186 4734–4743. (https://doi.org/10.4049/jimmunol.0904135)
Masters SL, Simon A, Aksentijevich I & Kastner DL 2009 Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. Annual Review of Immunology 27 621–668. (https://doi.org/10.1146/annurev.immunol.25.022106.141627)
O’Neill AS, van den Berg TK & Mullen GE 2013 Sialoadhesin – a macrophage-restricted marker of immunoregulation and inflammation. Immunology 138 198–207. (https://doi.org/10.1111/imm.12042)
Qi Y, Zhou Y, Chen X, Ye L, Zhang Q, Huang F, Cui B, Lin D, Ning G, Wang W, et al.2017 MicroRNA-4443 causes CD4+ T cells dysfunction by targeting TNFR-associated factor 4 in Graves’ disease. Frontiers in Immunology 8 1440–1440. (https://doi.org/10.3389/fimmu.2017.01440)
Rönnblom L & Eloranta ML 2013 The interferon signature in autoimmune diseases. Current Opinion in Rheumatology 25 248–253. (https://doi.org/10.1097/BOR.0b013e32835c7e32)
Rossol M, Kraus S, Pierer M, Baerwald C & Wagner U 2012 The CD14(bright) CD16+ monocyte subset is expanded in rheumatoid arthritis and promotes expansion of the Th17 cell population. Arthritis and Rheumatism 64 671–677. (https://doi.org/10.1002/art.33418)
Smith TJ & Hegedüs L 2016 Graves’ disease. New England Journal of Medicine 375 1552–1565. (https://doi.org/10.1056/NEJMra1510030)
Taylor PN, Albrecht D, Scholz A, Gutierrez-Buey G, Lazarus JH, Dayan CM & Okosieme OE 2018 Global epidemiology of hyperthyroidism and hypothyroidism. Nature Reviews: Endocrinology 14 301–316. (https://doi.org/10.1038/nrendo.2018.18)
Weider T, Richardson SJ, Morgan NG, Paulsen TH, Dahl-Jørgensen K & Hammerstad SS 2021 HLA Class I upregulation and antiviral immune responses in Graves disease. Journal of Clinical Endocrinology and Metabolism 106 1763–1774. (https://doi.org/10.1210/clinem/dgaa958)
Xiong YS, Zhou YH, Rong GH, Wu WL, Liang Y, Yang ZX, Geng HL & Zhong RQ 2009 Siglec-1 on monocytes is a potential risk marker for monitoring disease severity in coronary artery disease. Clinical Biochemistry 42 1057–1063. (https://doi.org/10.1016/j.clinbiochem.2009.02.026)
Xiong YS, Cheng Y, Lin QS, Wu AL, Yu J, Li C, Sun Y, Zhong RQ & Wu LJ 2014 Increased expression of Siglec-1 on peripheral blood monocytes and its role in mononuclear cell reactivity to autoantigen in rheumatoid arthritis. Rheumatology 53 250–259. (https://doi.org/10.1093/rheumatology/ket342)
Yin X, Sachidanandam R, Morshed S, Latif R, Shi R & Davies TF 2014 mRNA-Seq reveals novel molecular mechanisms and a robust fingerprint in Graves’ disease. Journal of Clinical Endocrinology and Metabolism 99 E2076–E2083. (https://doi.org/10.1210/jc.2014-1735)
York MR, Nagai T, Mangini AJ, Lemaire R, van Seventer JM & Lafyatis R 2007 A macrophage marker, siglec-1, is increased on circulating monocytes in patients with systemic sclerosis and induced by type I interferons and toll-like receptor agonists. Arthritis and Rheumatism 56 1010–1020. (https://doi.org/10.1002/art.22382)
Ziegler-Heitbrock L & Hofer TP 2013 Toward a refined definition of monocyte subsets. Frontiers in Immunology 4 23. (https://doi.org/10.3389/fimmu.2013.00023)
Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, Leenen PJM, Liu YJ, MacPherson G, Randolph GJ, et al.2010 Nomenclature of monocytes and dendritic cells in blood. Blood 116 e74–e80. (https://doi.org/10.1182/blood-2010-02-258558)
