Interleukins and Some Physiological Parameters in Patients with Autoimmune Thyroid Disorders

The prevalence and incidence of autoimmune thyroid diseases (AITD), presenting as Graves’ disease (GD) or Hashimoto’s thyroiditis (HT), has increased significantly in recent decades. It is crucial to identify immunological and pathomorphological factors involved in thyroid autoimmunity. Classically, HT has long been considered as a T helper (Th)1-mediated disease, while a Th2-driven autoimmune response is dominant for GD. Recently, Th17 cells have been established to play a role in the pathogenesis of AITD, however, their contribution to the initiation and progression of AITD remains unclear. Furthermore, selenium deficiency can impair the differentiation of Th cells, leading to dysfunction of cellular and humoral response. The aim of this thesis was to explore the role of Th17 cells in the pathogenesis of HT and GD by the use of different morphology methods and xMAP technology, and correlating these data with the selenium status. The initial study included 29 adult patients with AITD who underwent thyroidectomy, whereas subsequent clinical research project involved 52 patients with newly diagnosed, treatment-naïve AITD, as well as 26 healthy subjects served as controls. The plasma levels of Th17-associated cytokines – interleukin (IL)-17, IL-22, IL-23, IL-6, and IL-10 and the distribution and levels of immunoexpression IL-17, IL-23, and IL-1β within thyroid tissue were measured to characterize Th17 immune response in AITD. The integrity of the thyroid follicle by studying immunoexpression of cellular tight junctions – zonula occludens-1 and claudin-1 proteins, coupled to IL-17 and CD68, was explored. In addition, the selenium status was assessed. No significant differences in the plasma levels of Th17-associated cytokines were found among the patients with AITD and control subjects. However, the expression level of IL-17 in the thyrocytes was significantly higher in the HT and GD patients than in controls, simultaneously correlating with IL-23 and IL-1β immunopositivity in the HT group. Plasma Th17-associated cytokines’ levels were positively correlated with the severity of hyperthyroidism, independent of autoantibody levels, thus suggesting their possible role in GD pathogenesis. The changes in molecules of thyrocyte junctional complexes highlighting impairment of the integrity of thyroid follicle in HT were observed, but no significant association with IL-17 was found. Although no difference in selenium levels was observed between the AITD patients and controls, the results of the given research suggest the selenium status of the Latvian patients with newly diagnosed GD or HT is at a suboptimal level. Plasma selenium levels were negatively correlated with anti-thyroperoxidase (TPO) autoantibody titres in the HT patients, thus supporting the immunomodulatory role of selenium in AITD. Moreover, HT patients with higher anti-TPO autoantibody levels had lower levels of selenium, suggesting that these patients might benefit from selenium supplementation. Essential information deepening our knowledge about thyroid autoimmunity was obtained conducting this research, however, further experimental studies exploring the role and regulatory effects of Th17-related cytokines in the pathogenesis of AITD are required. More data from clinical studies are needed for a better understanding of the relationship between selenium supplementation and immune response.

The prevalence and incidence of autoimmune thyroid diseases (AITD), presenting as Graves’ disease (GD) or Hashimoto’s thyroiditis (HT), has increased significantly in recent decades. It is crucial to identify immunological and pathomorphological factors involved in thyroid autoimmunity. Classically, HT has long been considered as a T helper (Th)1-mediated disease, while a Th2-driven autoimmune response is dominant for GD. Recently, Th17 cells have been established to play a role in the pathogenesis of AITD, however, their contribution to the initiation and progression of AITD remains unclear. Furthermore, selenium deficiency can impair the differentiation of Th cells, leading to dysfunction of cellular and humoral response. The aim of this thesis was to explore the role of Th17 cells in the pathogenesis of HT and GD by the use of different morphology methods and xMAP technology, and correlating these data with the selenium status. The initial study included 29 adult patients with AITD who underwent thyroidectomy, whereas subsequent clinical research project involved 52 patients with newly diagnosed, treatment-naïve AITD, as well as 26 healthy subjects served as controls. The plasma levels of Th17-associated cytokines – interleukin (IL)-17, IL-22, IL-23, IL-6, and IL-10 and the distribution and levels of immunoexpression IL-17, IL-23, and IL-1β within thyroid tissue were measured to characterize Th17 immune response in AITD. The integrity of the thyroid follicle by studying immunoexpression of cellular tight junctions – zonula occludens-1 and claudin-1 proteins, coupled to IL-17 and CD68, was explored. In addition, the selenium status was assessed. No significant differences in the plasma levels of Th17-associated cytokines were found among the patients with AITD and control subjects. However, the expression level of IL-17 in the thyrocytes was significantly higher in the HT and GD patients than in controls, simultaneously correlating with IL-23 and IL-1β immunopositivity in the HT group. Plasma Th17-associated cytokines’ levels were positively correlated with the severity of hyperthyroidism, independent of autoantibody levels, thus suggesting their possible role in GD pathogenesis. The changes in molecules of thyrocyte junctional complexes highlighting impairment of the integrity of thyroid follicle in HT were observed, but no significant association with IL-17 was found. Although no difference in selenium levels was observed between the AITD patients and controls, the results of the given research suggest the selenium status of the Latvian patients with newly diagnosed GD or HT is at a suboptimal level. Plasma selenium levels were negatively correlated with anti-thyroperoxidase (TPO) autoantibody titres in the HT patients, thus supporting the immunomodulatory role of selenium in AITD. Moreover, HT patients with higher anti-TPO autoantibody levels had lower levels of selenium, suggesting that these patients might benefit from selenium supplementation. Essential information deepening our knowledge about thyroid autoimmunity was obtained conducting this research, however, further experimental studies exploring the role and regulatory effects of Th17-related cytokines in the pathogenesis of AITD are required. More data from clinical studies are needed for a better understanding of the relationship between selenium supplementation and immune response.

Pathogenesis of autoimmune thyroid disease (ATD) is multifactorial. Helicobacter pylori (Hp) infection has been proposed to be involved in nongastrointestinal conditions and reported more frequently in ATD adult patients. We evaluated the prevalence of Hp antibodies in young ATD patients and investigated the possibility that a susceptible immunogenetic profile could influence the development of ATD in subjects with Hp infection. We retrospectively studied 90 children with ATD (median age 11.2 yr), 70 age- and sex-matched healthy subjects as controls, and 65 patients with Turner syndrome (median age 18.8 yr). Antibodies to Hp were determined at diagnosis in ATD patients and, in Turner patients, at the last control in cases without ATD and before the appearance of thyroid autoantibodies in the others. Serological and molecular human leukocyte antigen (HLA) typing for classes I and II polymorphisms was performed. Prevalence of positive Hp serology resulted significantly higher in ATD patients than controls (P = 0.032). No association was found between individual HLA alleles and Hp serology. HLA-A1, B8, and DRB1*0301 were found significantly associated with ATD. A significant interaction between HLA-DRB1*0301 and Hp infection was present in ATD patients and not controls (P = 0.007), suggesting that the copresence of these two factors might favor ATD development. A similar phenomenon was observed in Turner syndrome patients (P = 0.02; cumulative Mantel test, P = 0.0001). Another target of Hp-elicited immune inflammatory response might be the thyroid gland in subjects with a peculiar immunogenetic profile so that ATD may be a consequence. Our findings suggest the opportunity of eradicating Hp infection in children with ATD and/or susceptible HLA alleles.

Autoimmune thyroid disease (AITD) is an organ-specific disorder due to the interplay between environmental and genetic factors. Toll-like receptors (TLRs) are pattern recognition receptors expressed abundantly on monocytes. There is a paucity of data on TLR expression in AITD. The aim of this study was to examine TLR expression, activation, ligands, and downstream signaling adaptors in peripheral blood mononuclear cells (PBMCs) extracted from untreated AITD patients and healthy controls. We isolated PBMC of 30 healthy controls, 36 patients with untreated Hashimoto's thyroiditis, and 30 patients with newly onset Graves' disease. TLR mRNA, protein expression, TLR ligands, and TLR adaptor molecules were measured using real-time PCR, Western blot, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). PBMC was simulated with TLR agonists. The effects of TLR agonists on the viability of human PBMC were evaluated using the MTT assay. The supernatants of cell cultures were measured for the pro-inflammatory cytokines, interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), and IL-10 by ELISA. TLR2, TLR3, TLR9, and TLR10 mRNA were significantly increased in AITD patients compared with controls. TLR2, TLR3, TLR9, high mobility group box 1 (HMGB1), and RAGE expression on monocytes was higher in patients than control at baseline and TLR agonists' stimulation. The release of TNF-α and IL-6 was significantly increased in PBMCs from AITD patients with TLR agonists, while IL-10 was significantly decreased. Downstream targets of TLR, myeloid differentiation factor 88 (MyD88), and myeloid toll/IL-1 receptor-domain containing adaptor-inducing interferon-β were significantly elevated in AITD patients. Levels of TLR2 ligands, HMGB1, and heat shock protein 60 were significantly elevated in AITD patients compared with those in controls and positively correlated with TgAb and TPOAb, while sRAGE concentration was significantly decreased in AITD patients. This work is the first to show that TLR2, TLR3, and TLR9 expression and activation are elevated in the PBMCs of patients with AITD and TLRs may participate in the pathogenesis of AITD.

Graves' disease (GD) and Hashimoto's disease (HD) are well known autoimmune thyroid diseases (AITDs), and the severity and intractability of AITDs varies among patients. Thyroid peroxidase (TPO) is a thyroid-specific antigen. The levels of anti-thyroid peroxidase antibody (TPOAb) were higher in patients with HD and may be associated with thyroid destruction. In this study, we genotyped eight single nucleotide polymorphisms (SNPs) in the TPO gene to clarify the association of TPO gene polymorphisms with the development, severity and intractability of AITD. We genotyped TPO rs2071399G/A, rs2071400C/T, rs2071402A/G, rs2071403A/G, rs1126799C/T, rs1126797T/C, rs732609A/C, and rs2048722A/G polymorphisms in 145 patients with GD, 147 patients with HD and 92 healthy controls by PCR-RFLP method. TPO rs2071400 T carriers (CT + TT genotypes) were more frequent in AITD, GD, and HD patients (p=0.0079, 0.0041, and 0.0488, respectively). The TPO rs2071403 GG genotype was more frequent in AITD, GD, and HD patients (p=0.0227, 0.0465, and 0.0305, respectively). There was no significant association between the SNPs and the prognosis of AITD. Serum levels of TPOAb were significantly higher in AITD patients with TPO rs2071400 T carriers (CT + TT genotypes) than in those with the CC genotype (p=0.0295), and were also significantly higher in AITD patients with TPO rs2048722 T carriers (CT + TT genotypes) than in those with the CC genotype (p=0.0056). In conclusion, TPO rs2071400 and rs2071403 polymorphisms were associated with the development of HD and GD, but not with the prognosis. Moreover, TPO rs2071400 and rs2048722 polymorphisms were associated with the serum levels of TPOAb.

In recent years, the emergence of multiplex technology that can simultaneously measure multiple anti-islet autoantibodies has become particularly valuable for the staging and early diagnosis of immune-mediated type 1 diabetes (T1D). While it has been established that 20%-30% of T1D patients suffer from autoimmune thyroid disease (AITD), there is limited available data regarding the presence of anti-islet autoantibodies in AITD patients. Among commercially available anti-islet autoantibodies, glutamic acid decarboxylase 65 autoantibodies (GADAs) are often the first marker measured in general clinical practice. To investigate the frequency of anti-islet autoantibodies in AITD patients. Our study involved four hundred ninety-five AITD patients, categorized into three distinct groups: AITD with T1D (n = 18), AITD with phenotypic type 2 diabetes (T2D) (n = 81), and AITD without diabetes (n = 396), and the enzyme-linked immunosorbent assay (ELISA) was employed to determine the frequencies of 3 Screen Islet Cell Autoantibody (3 Screen ICA), GADA, insulinoma-associated antigen-2 autoantibodies (IA-2As), and zinc transporter 8 autoantibodies (ZnT8As) within these groups. The frequency of 3 Screen ICA in AITD patients with T1D, T2D, and those without diabetes were 88.9%, 6.2%, and 5.1%, respectively, with no significant difference seen between the latter two groups. Notably, the frequency of 3 Screen ICA was 11.1% higher in AITD patients with T1D, 1.3% higher in AITD patients with T2D, and 1.1% higher in AITD patients without diabetes compared to GADA, respectively. Furthermore, 12.5%, 20.0%, and 20.0% of the 3 Screen ICA-positive patients were negative for GADA. Additionally, 1.3% of the AITD patients who tested negative for 3 Screen ICA in both the AITD with T2D and non-diabetic AITD groups were found to be positive for individual autoantibodies. Among the 3 Screen ICA-positive patients, there was a significantly higher proportion of individuals with multiple autoantibodies in AITD patients with T1D compared to those without diabetes (37.5% vs 5.0%, P < 0.05). However, this proportion was similar to that in AITD patients with T2D (20.0%). Nevertheless, there was no significant difference in 3 Screen ICA titers between AITD patients with T1D and those without diabetes (436.8 ± 66.4 vs 308.1 ± 66.4 index). Additionally, no significant difference in 3 Screen ICA titers was observed between Graves' disease and Hashimoto's thyroiditis in any of the groups. Our findings reveal that some AITD patients without diabetes exhibit 3 Screen ICA titers comparable to those in AITD patients with T1D. Thus, 3 Screen ICA outperforms GADA in identifying latent anti-islet autoantibody-positive individuals among AITD patients.

Transmembrane protein 39A (TMEM39A) gene polymorphisms have been related to various autoimmune diseases, but the relationship between TMEM39A polymorphisms and autoimmune thyroid disease (AITD) remains unknown. This study was aimed at investigating whether the polymorphisms of the TMEM39A were associated with AITD in the Chinese Han population. A case-control study was performed in a total of 906 AITD patients and 744 healthy controls. Four single nucleotide polymorphisms, including rs1132200, rs12492609, rs2282175, and rs7629750, in TMEM39A were examined by polymerase chain reaction-ligase detection reaction. We found that the allele T of rs12492609 in TMEM39A was associated with AITD and Hashimoto's thyroiditis (HT) (p = 0.023; p = 0.028 respectively). Compared with controls, the frequency of haplotype CCA in AITD patients was higher than that in controls (p = 0.036), but the frequency of haplotype CTA in AITD and HT patients was lower than that in controls (p = 0.046; p = 0.047 respectively). In addition, the frequency of allele A at rs7629750 in AITD patients with onset age ≤18 years old was higher than that in AITD patients with onset age ≥19 (p = 0.046). Further, there was an obvious difference in the genotype distributions of rs12492609 and rs7629750 between HT patients with hypothyroidism and those without hypothyroidism (p = 0.034 and p = 0.023, respectively). Our study first reveals that the polymorphisms of the TMEM39A gene are associated with the susceptibility to AITD, especially for early-onset AITD and HT with hypothyroidism.

Autoimmune thyroid diseases (AITD) are a group of both B cell- and T cell-mediated organ-specific autoimmune diseases. Graves’ disease and Hashimoto thyroiditis are the two main clinical presentations of AITD. Both genetic and environmental factors have important roles in the development of AITD. Epigenetics have been considered to exert key roles in integrating those genetic and environmental factors, and epigenetic modifications caused by environmental factors may drive genetically susceptibility individuals to develop AITD. Recent studies on the epigenetics of AITD have provided some novel insights into the pathogenesis of AITD. The aim of this review is to provide an overview of recent advances in the epigenetic mechanisms of AITD, such as DNA methylation, histone modifications, and non-coding RNAs. This review highlights the key roles of epigenetics in the pathogenesis of AITD and potential clinical utility. However, the epigenetic roles in AITD are still not fully elucidated, and more researches are needed to provide further deeper insights into the roles of epigenetics in AITD and to uncover new therapeutic targets. Although there are many studies assessing the epigenetic modifications in AITD patients, the clinical utility of epigenetics in AITD remains poorly defined. More studies are needed to identify the underlying epigenetic modifications that can contribute to accurate diagnosis of AITD, adequate choice of treatment approach, and precise prediction of treatment outcomes.

Graves disease and Hashimoto disease form part of the spectrum of autoimmune thyroid disease (AITD), to which genetic and environmental factors are recognized contributors. Epigenetics provides a potential link between environmental influences, gene expression, and thyroid autoimmunity. DNA methylation (DNAm) is the best studied epigenetic process, and global hypomethylation of leukocyte DNA is reported in several autoimmune disorders. This review summarizes the current understanding of DNAm in AITD. Targeted DNAm studies of blood samples from AITD patients have reported differential DNAm in the promoter regions of several genes implicated in AITD, including TNF, IFNG, IL2RA, IL6, ICAM1, and PTPN22. In many cases, however, the findings await replication and are unsupported by functional studies to support causal roles in AITD pathogenesis. Furthermore, thyroid hormones affect DNAm, and in many studies confounding by reverse causation has not been considered. Recent studies have shown that DNAm patterns in candidate genes including ITGA6, PRKAA2, and DAPK1 differ between AITD patients from regions with different iodine status, providing a potential mechanism for associations between iodine and AITD. Research focus in the field is moving from candidate gene studies to an epigenome-wide approach. Genome-wide methylation studies of AITD patients have demonstrated multiple differentially methylated positions, including some in immunoregulatory genes such as NOTCH1, HLA-DRB1, TNF, and ICAM1. Large, epigenome-wide studies are required to elucidate the pathophysiological role of DNAm in AITD, with the potential to provide novel diagnostic and prognostic biomarkers as well as therapeutic targets.

Myeloid related proteins are up-regulated in autoimmune thyroid diseases and activate toll-like receptor 4 and pro-inflammatory cytokines in vitro

The aim of this study was to investigate the association between signal transducer and activator of transcription 3 (STAT3) polymorphisms and autoimmune thyroid diseases and clinical features. We genotyped six single-nucleotide polymorphisms (SNPs) rs1053005, rs2293152, rs744166, rs17593222, rs2291281, and rs2291282 of STAT3 gene in 667 patients with autoimmune thyroid disease (417 Graves’ disease (GD) and 250 Hashimoto’s thyroiditis (HT)) and 301 healthy controls. The allele A from rs1053005 was significantly less frequent in both GD and HT patients (P = 0.0024, OR = 0.6958, 95%CI = 0.5508–0.8788; P = 0.0091, OR = 0.7013, 95%CI = 0.5397–0.9112, respectively). The AA genotype of rs1053005 was less in GD and HT patients too (P = 0.0025,OR = 0.6278, 95%CI = 0.466–0.847) and (P = 0.0036,OR = 0.601, 95%CI = 0.428–0.843). The allele G from rs17593222 increased the susceptibility to the ophthalmopathy development both in autoimmune thyroid disease (AITD) and GD patients (P = 0.0007, OR = 3.980, 95%CI = 1.871–8.464; P = 0.0081, OR = 3.378, 95%CI = 1.441–7.919, respectively). The allele A and AA genotype of SNP rs1053005 may protect individuals from the susceptibility to AITD and their frequency decreased in AITD patients. In addition, the allele G of rs17593222 may increase the ophthalmopathy risk in AITD patients. Our findings suggest the existence of association between STAT3 gene and AITD, thus adding STAT3 gene to the list of the predisposing genes to AITD.

Follicular helper T (Tfh) cells exert an important role in the autoimmune diseases. Our study aimed to explore the role of Tfh cells in patients with autoimmune thyroid disease (AITD). Tfh cell is a new subset regulating the antibody production of B cell. Previous studies implicated CD4+CXCR5+ICOShigh or CD4+CXCR5+PD-1high as the markers of circulating Tfh cells. Sixty-five patients with AITD and 30 healthy controls were enrolled in the current study. The percentages of circulating Tfh cells were assessed by flow cytometry. The correlation between the percentages of CD4+CXCR5+ICOShigh T cells and the levels of autoantibodies or hormones was also analyzed. Additionally, polyphasic methods were applied to investigate the status of Tfh cells in thyroid glands of Hashimoto's thyroiditis patients. Increased percentages of circulating Tfh cells in AITD patients were detected, and a positive correlation between the percentages of circulating Tfh cells and the serum concentrations of anti-TSH receptor-Ab/thyroperoxidase-Ab/thyroglobulin-Ab was confirmed. A positive or modest relationship between the percentages of circulating Tfh cells and serum free T3 or free T4 was revealed in Graves' disease patients. Additionally, follow-up analysis indicated that in some Graves' disease patients the percentage of circulating Tfh cells decreased after treatment. Furthermore, a certain number of CD4+CXCR5+ICOShigh T cells together with enhanced expression of IL-21 and Bcl-6 mRNA were detected in thyroid tissues from Hashimoto's thyroiditis patients. The current study discovered an increased frequency of Tfh cells in AITD patients, which implies that this cell subset might play an important role in the pathogenesis of AITD.

IntroductionPatients with autoimmune thyroid disease (AITD) show defects in their immune-regulatory mechanisms. Herein we assessed the expression and function of galectin-1 and galectin-9 (Gal-1, Gal-9) in dendritic cells (DCs) from patients with AITD.Materials and MethodsPeripheral blood samples from 25 patients with Graves’ disease (GD), 11 Hashimoto’s thyroiditis (HT), and 24 healthy subjects were studied. Thyroid tissue samples from 44 patients with AITD and 22 patients with goiter were also analyzed. Expression and function of Gal-1 and Gal-9 was assessed by quantitative RT-PCR, immunofluorescence and flow cytometry.ResultsA diminished expression of Gal-9, but not of Gal-1, by peripheral blood DCs was observed in GD patients, mainly in those with Graves´ ophthalmopathy, and a significant negative association between disease severity and Gal-9 expression was detected. In addition, the mRNA levels of Gal-9 and its ligand TIM-3 were increased in thyroid tissue from AITD patients and its expression was associated with the levels of Th1/Th12/Th17 cytokines. Immunofluorescence studies proved that intrathyroidal Gal-9 expression was confined to DCs and macrophages. Finally, in vitro functional assays showed that exogenous Gal-9 had a suppressive effect on the release of Th1/Th2/Th17 cytokines by DC/lymphocyte autologous co-cultures from both AITD patients and healthy controls.ConclusionsThe altered pattern of expression of Gal-9 in peripheral blood DCs from GD patients, its correlation with disease severity as well as its ability to suppress cytokine release suggest that Gal-9 could be involved in the pathogenesis of AITD.

Th1 or Th2 cytokines are correlated with Tregs and T cell subsets and pregnancy outcomes in patients with autoimmune thyroid disease during early, middle, late pregnancy, and postpartum period

THU0340 Cytokines’ study in patients with different subtypes of juvenile idiopathic arthritis