[Show abstract][Hide abstract] ABSTRACT: We previously showed that an HLA-DR variant containing arginine at position 74 of the DRβ1 chain (DRβ1-Arg74) is the specific
HLA class II variant conferring risk for autoimmune thyroid diseases (AITD). We also identified 5 thyroglobulin (Tg) peptides
that bound to DRβ1-Arg74. We hypothesized that blocking the binding of these peptides to DRβ1-Arg74 could block the continuous
T-cell activation in thyroiditis needed to maintain the autoimmune response to the thyroid. The aim of the current study was
to identify small molecules that can block T-cell activation by Tg peptides presented within DRβ1-Arg74 pockets. We screened
a large and diverse library of compounds and identified one compound, Cepharanthine that was able to block peptide binding
to DRβ1-Arg74. We then showed that Tg.2098 is the dominant peptide when inducing experimental autoimmune thyroiditis (EAT)
in NOD mice expressing human DRβ1-Arg74. Furthermore, Cepharanthine blocked T-cell activation by thyroglobulin peptides,
in particular Tg.2098 in mice that were induced with EAT. For the first time we identified a small molecule that can block
Tg peptide binding and presentation to T-cells in autoimmune thyroiditis. If confirmed Cepharanthine could potentially have
a role in treating human AITD.
Preview · Article · Dec 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: We conducted linkage analysis to follow up earlier work on microvascular complications of type 1 diabetes (T1D). We analyzed 415 families (2,008 individuals) previously genotyped for 402 SNP markers spanning chromosome 6. We did linkage analysis for the phenotypes of retinopathy and nephropathy. For retinopathy, two linkage peaks were mapped: one located at the HLA region and another novel locus telomeric to HLA. For nephropathy, a linkage peak centromeric to HLA was mapped, but the linkage peak telomeric to HLA seen in retinopathy was absent. Because of the strong association of T1D with DRB1
03:01 and DRB1
04:01, we stratified our analyses based on families whose probands were positive for DRB1
03:01 or DRB1
04:01. When analyzing the DRB1
03:01-positive retinopathy families, in addition to the novel telomeric locus, one centromeric to HLA was identified at the same location as the nephropathy peak. When we stratified on DRB1
04:01-positive families, the HLA telomeric peak strengthened but the centromeric peak disappeared. Our findings showed that HLA and non-HLA loci on chromosome 6 are involved in T1D complications’ expression. While the HLA region is a major contributor to the expression of T1D, our results suggest an interaction between specific HLA alleles and other loci that influence complications’ expression.
Full-text · Article · Oct 2015 · Journal of Diabetes Research
[Show abstract][Hide abstract] ABSTRACT: Diabetes and hepatitis C infection are both prevalent diseases worldwide, and are associated with increased morbidity and mortality. Most studies, but not all, have shown that patients with chronic hepatitis C are more prone to develop type 2 diabetes (T2D) compared to healthy controls, as well as when compared to patients with other liver diseases, including hepatitis B. Furthermore, epidemiological studies have revealed that patients with T2D may also be at higher risk for worse outcomes of their hepatitis C infection, including reduced rate of sustained virological response, progression to fibrosis and cirrhosis, and higher risk for development of hepatocellular carcinoma. Moreover, hepatitis C infection and mainly its treatment, interferon α, can trigger the development of type 1 diabetes. In this review, we discuss the existing data on this two-way association between diabetes and hepatitis C infection with emphasis on possible mechanisms. It remains to be determined whether the new curative therapies for chronic hepatitis C will improve outcomes in diabetic hepatitis C patients, and conversely whether treatment with Metformin will reduce complications from hepatitis C virus infection. We propose an algorithm for diabetes screening and follow-up in hepatitis C patients.
Full-text · Article · Oct 2015 · Frontiers in Endocrinology
[Show abstract][Hide abstract] ABSTRACT: Both environmental and genetic triggers factor into the etiology of autoimmune thyroid disease (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT). Although the exact pathogenesis and causative interaction between environment and genes are unknown, GD and HT share similar immune-mediated mechanisms of disease. They both are characterized by the production of thyroid autoantibodies and by thyroidal lymphocytic infiltration, despite being clinically distinct entities with thyrotoxicosis in GD and hypothyroidism in HT. Family and population studies confirm the strong genetic influence and inheritability in the development of AITD. AITD susceptibility genes can be categorized as either thyroid specific (Tg, TSHR) or immune-modulating (FOXP3, CD25, CD40, CTLA-4, HLA), with HLA-DR3 carrying the highest risk. Of the AITD susceptibility genes, FOXP3 and CD25 play critical roles in the establishment of peripheral tolerance while CD40, CTLA-4, and the HLA genes are pivotal for T lymphocyte activation and antigen presentation. Polymorphisms in these immune-modulating genes, in particular, significantly contribute to the predisposition for GD, HT and, unsurprisingly, other autoimmune diseases. Emerging evidence suggests that single nucleotide polymorphisms (SNPs) in the immunoregulatory genes may functionally hinder the proper development of central and peripheral tolerance and alter T cell interactions with antigen presenting cells (APCs) in the immunological synapse. Thus, susceptibility genes for AITD contribute directly to the key mechanism underlying the development of organ-specific autoimmunity, namely the breakdown in self-tolerance. Here we review the major immune-modulating genes that are associated with AITD and their potential functional effects on thyroidal immune dysregulation.
Published by Elsevier Ltd.
No preview · Article · Jul 2015 · Journal of Autoimmunity
[Show abstract][Hide abstract] ABSTRACT: Autoimmune thyroid diseases (AITD) and Type 1 diabetes (T1D) frequently occur in the same individual pointing to a strong shared genetic susceptibility. Indeed, the co-occurrence of T1D and AITD in the same individual is classified as a variant of the autoimmune polyglandular syndrome type 3 (designated APS3v). Our aim was to identify new genes and mechanisms causing the co-occurrence of T1D + AITD (APS3v) in the same individual using a genome-wide approach. For our discovery set we analyzed 346 Caucasian APS3v patients and 727 gender and ethnicity matched healthy controls. Genotyping was performed using the Illumina Human660W-Quad.v1. The replication set included 185 APS3v patients and 340 controls. Association analyses were performed using the PLINK program, and pathway analyses were performed using the MAGENTA software. We identified multiple signals within the HLA region and conditioning studies suggested that a few of them contributed independently to the strong association of the HLA locus with APS3v. Outside the HLA region, variants in GPR103, a gene not suggested by previous studies of APS3v, T1D, or AITD, showed genome-wide significance (p < 5 × 10(-8)). In addition, a locus on 1p13 containing the PTPN22 gene showed genome-wide significant associations. Pathway analysis demonstrated that cell cycle, B-cell development, CD40, and CTLA-4 signaling were the major pathways contributing to the pathogenesis of APS3v. These findings suggest that complex mechanisms involving T-cell and B-cell pathways are involved in the strong genetic association between AITD and T1D.
Published by Elsevier Ltd.
No preview · Article · Apr 2015 · Journal of Autoimmunity
[Show abstract][Hide abstract] ABSTRACT: The autoimmune thyroid diseases (AITD) include a number of conditions which have in common cellular and humoral immune responses targeted at the thyroid gland. The AITD include Graves' disease (GD) and Hashimoto's thyroiditis (HT), both of which involve infiltration of the thyroid by T and B cells reactive with thyroid antigens, production of thyroid autoantibodies, with the resultant clinical manifestations (hyperthyroidism in GD and hypothyroidism in HT). This chapter points to the possibility of involvement of an infectious agent in the pathogenesis of. Several studies point to the involvement of bacterial and viral agents in the pathogenesis of Graves' and Hashimoto's diseases. Various mechanisms have been proposed to explain induction of autoimmunity by infection, but it seems that two possibilities may be important to thyroid autoimmunity-amely, molecular mimicry (perhaps to retroviruses) and MHC class II antigen induction. However, it should be remembered that the association between AITD and infections may be merely coincidental and not etiological.
[Show abstract][Hide abstract] ABSTRACT: Context:
Amiodarone (AMIO) is one of the most effective antiarrhythmic drugs available; however, its use is limited by a serious side effect profile, including thyroiditis. The mechanisms underlying AMIO thyroid toxicity have been elusive; thus, identification of novel approaches in order to prevent thyroiditis is essential in patients treated with AMIO.
Our aim was to evaluate whether AMIO treatment could induce endoplasmic reticulum (ER) stress in human thyroid cells and the possible implications of this effect in AMIO-induced destructive thyroiditis.
Here we report that AMIO, but not iodine, significantly induced the expression of ER stress markers including Ig heavy chain-binding protein (BiP), phosphoeukaryotic translation initiation factor 2α (eIF2α), CCAAT/enhancer-binding protein homologous protein (CHOP) and spliced X-box binding protein-1 (XBP-1) in human thyroid ML-1 cells and human primary thyrocytes. In both experimental systems AMIO down-regulated thyroglobulin (Tg) protein but had little effect on Tg mRNA levels, suggesting a mechanism involving Tg protein degradation. Indeed, pretreatment with the specific proteasome inhibitor MG132 reversed AMIO-induced down-regulation of Tg protein levels, confirming a proteasome-dependent degradation of Tg protein. Corroborating our findings, pretreatment of ML-1 cells and human primary thyrocytes with the chemical chaperone 4-phenylbutyric acid completely prevented the effect of AMIO on both ER stress induction and Tg down-regulation.
We identified ER stress as a novel mechanism contributing to AMIO-induced destructive thyroiditis. Our data establish that AMIO-induced ER stress impairs Tg expression via proteasome activation, providing a valuable therapeutic avenue for the treatment of AMIO-induced destructive thyroiditis.
No preview · Article · Oct 2014 · Journal of Clinical Endocrinology & Metabolism
[Show abstract][Hide abstract] ABSTRACT: Graves disease (GD) is an autoimmune condition caused by interacting genetic and environmental factors. Genetic studies have mapped several single-nucleotide polymorphisms (SNPs) that are strongly associated with GD, but the mechanisms by which they trigger disease are unknown. We hypothesized that epigenetic modifications induced by microenvironmental influences of cytokines can reveal the functionality of GD-associated SNPs. We analyzed genome-wide histone H3 lysine 4 methylation and gene expression in thyroid cells induced by IFNα, a key cytokine secreted during viral infections, and overlapped them with known GD-associated SNPs. We mapped an open chromatin region overlapping two adjacent GD-associated SNPs (rs12101255 and rs12101261) in intron 1 of the thyroid stimulating hormone receptor (TSHR) gene. We then demonstrated that this region functions as a regulatory element through binding of the transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) at the rs12101261 site. Repression by PLZF depended on the rs12101261 disease susceptibility allele and was increased by IFNα. Intrathymic TSHR expression was decreased in individuals homozygous for the rs12101261 disease-associated genotype compared with carriers of the disease-protective allele. Our studies discovered a genetic-epigenetic interaction involving a noncoding SNP in the TSHR gene that regulates thymic TSHR gene expression and facilitates escape of TSHR-reactive T cells from central tolerance, triggering GD.
Preview · Article · Aug 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Context: Genetic and environmental factors play an essential role in the pathogenesis of Graves' disease (GD). Children with GD have less exposure time to environmental factors and therefore are believed to harbor stronger genetic susceptibility than adults. Objective: The aim of the study was to identify susceptibility loci that predispose to GD in young age of onset (AO) GD patients. Design and Participants: One hundred and six patients with young AO GD (onset <30 years-old) and 855 healthy subjects were studied. Cases and controls were genotyped using the Illumina infinium immunochip, designed to genotype 196,524 polymorphisms. Case control association analyses were performed using the PLINK computer package. Ingenuity Pathway Analysis program was used to carry out pathway analyses. Results: Immunochip genetic association analysis identified 30 single-nucleotide polymorphisms in several genes that were significantly associated with young AO GD, including MHC class I and class II genes, BTNL2, NOTCH4, TNFAIP3, and CXCR4. Candidate gene analysis revealed that most of the genes previously shown to be associated with adult-onset GD were also associated with young AO GD. Pathway analysis demonstrated that antigen presentation, T-helper cell differentiation and B-cell development were the major pathways contributing to the pathogenesis of young AO GD. Conclusions: Genetic analysis identified novel susceptibility loci in young AO GD adding a new dimension to the understanding of GD etiology.
Full-text · Article · Mar 2014 · The Journal of Clinical Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Recent advances in our understanding of genetic-epigenetic interactions have unraveled new mechanisms underlying the etiology of complex autoimmune diseases. Autoimmune thyroid diseases (AITDs) are highly prevalent, affecting 1% to 5% of the population. The major AITDs include Graves disease (GD) and Hashimoto's thyroiditis (HT); although these diseases contrast clinically, their pathogenesis involves shared immunogenetic mechanisms. Genetic data point to the involvement of both shared and unique genes. Among the shared susceptibility genes, HLA-DRβ1-Arg74 (human leukocyte antigen DR containing an arginine at position β74) confers the strongest risk. Recent genome-wide analyses have revealed new putative candidate genes. Epigenetic modulation is emerging as a major mechanism by which environmental factors interact with AITD susceptibility genes. Dissecting the genetic-epigenetic interactions underlying the pathogenesis of AITD is essential to uncover new therapeutic targets.
No preview · Article · Jan 2014 · Annual Review of Pathology Mechanisms of Disease
[Show abstract][Hide abstract] ABSTRACT: Graves disease (GD) and Hashimoto thyroiditis (HT) are two autoimmune disorders that commonly affect the thyroid gland. Thyroid peroxidase (TPO) and thyroglobulin (Tg) are two key thyroid antigens targeted by the immune system in autoimmune thyroid disease (AITD). The TPO antibodies are found in the majority of GD and HT patients and were recently found to react with two immunodominant epitopes located on the overlapping A and B domains. TPO antibodies recognize mostly conformational epitopes that depend on the structure and folding of the protein. Tg antibodies are less sensitive and specific than TPO antibodies in GD and HT. However, the Tg gene is one of the major autoimmune thyroid disease (AITD) susceptibility genes and is believed to be the key antigen-triggering AITD. Tg antigenicity may be influenced by post-translational modifications. It has been suggested that epitope spreading from Tg to TPO may occur in AITD, with Tg being the primary antigen responsible for breaking T- and B-cell tolerance and TPO being responsible for the maintenance of autoimmunity in AITD.
[Show abstract][Hide abstract] ABSTRACT: Type 1 diabetes (T1D) shows ∼40% concordance rate in monozygotic twins (MZ) suggesting a role for environmental factors and/or epigenetic modifications in the etiology of the disease. The aim of our study was to dissect the contribution of epigenetic factors, particularly, DNA methylation (DNAm), to the incomplete penetrance of T1D. We performed DNAm profiling in lymphocyte cell lines from 3 monozygotic (MZ) twin pairs discordant for T1D and 6 MZ twin pairs concordant for the disease using HumanMethylation27 BeadChip. This assay assesses the methylation state of 27,578 CpG sites, mostly located within proximal promoter regions. We identified 88 CpG sites displaying significant methylation changes in all T1D-discordant MZ twin pairs. Functional annotation of the genes with distinct CpG methylation profiles in T1D samples showed differential DNAm of immune response and defense response pathways between affected and unaffected twins. Integration of DNAm data with GWAS data mapped several known T1D associated genes, HLA, INS, IL-2RB, CD226, which showed significant differences in DNAm between affected and unaffected of twins. Our findings suggest that abnormalities of DNA methylation patterns, known to regulate gene transcription, may be involved in the pathogenesis of T1D.
No preview · Article · Nov 2013 · Journal of Autoimmunity
[Show abstract][Hide abstract] ABSTRACT: Autoimmune thyroid diseases (AITD) have become increasingly recognized as a complication of interferon-alpha (IFNα) therapy in patients with chronic Hepatitis C virus (HCV) infection. Interferon-induced thyroiditis (IIT) can manifest as clinical thyroiditis in approximately 15% of HCV patients receiving IFNα and subclinical thyroiditis in up to 40% of patients, possibly resulting in either dose reduction or discontinuation of IFNα treatment. However, the exact mechanisms that lead to the development of IIT are unknown and may include IFNα-mediated immune-recruitment as well as direct toxic effects on thyroid follicular cells. We hypothesized that IIT develops in genetically predisposed individuals whose threshold for developing thyroiditis is lowered by IFNα. Therefore, our aim was to identify the susceptibility genes for IIT. We used a genomic convergence approach combining genetic association data with transcriptome analysis of genes upregulated by IFNα. Integrating results of genetic association, transcriptome data, pathway, and haplotype analyses enabled the identification of 3 putative loci, SP100/110/140 (2q37.1), HLA (6p21.3), and TAP1 (6p21.3) that may be involved in the pathogenesis of IIT. Immune-regulation and apoptosis emerged as the predominant mechanisms underlying the etiology of IIT.
No preview · Article · May 2013 · Journal of Autoimmunity
[Show abstract][Hide abstract] ABSTRACT: Context:
TSH receptor antibodies (TRAb) cause Graves' disease (GD) hyperthyroidism. Widely available TRAb measurement methods have been significantly improved recently. However, the role of TRAb measurement in the differential diagnosis of hyperthyroidism, the prediction of remission of GD hyperthyroidism, the prediction of fetal/neonatal thyrotoxicosis, and the clinical assessment of Graves' ophthalmopathy (GO) are controversial.
We reviewed and analyzed the literature reporting primary data on the clinical use of TRAb. We focused our analyses on clinical studies analyzing third-generation TRAb assays.
The performance of TRAb in the differential diagnosis of overt hyperthyroidism is excellent, with sensitivity and specificity in the upper 90%. TRAb can accurately predict short-term relapses of hyperthyroidism after a course of antithyroid drugs but are less effective in predicting long-term relapses or remissions. Pregnancies in women with GD with negative TRAb are highly unlikely to result in fetal hyperthyroidism, whereas high titers of TRAb in pregnancy require careful fetal monitoring. GD patients with GO frequently have high TRAb levels. However, there are insufficient data to use the test to predict the clinical course of GO and response to treatment.
Third-generation TRAb assays are suitable in the differential diagnosis of hyperthyroidism. In GD, TRAb should be tested before deciding whether methimazole can be stopped. TRAb should be used in pregnant women with GD to assess the risk of fetal thyrotoxicosis. The use of TRAb in GO requires further studies.
Preview · Article · Mar 2013 · The Journal of Clinical Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Although HLA alleles are associated with type 1 diabetes, association with microvascular complications remains controversial. We tested HLA association with complications in multiplex type 1 diabetes families. Probands from 425 type 1 diabetes families from the Human Biological Data Interchange (HBDI) collection were analyzed. The frequencies of specific HLA alleles in patients with complications were compared with the frequencies in complications-free patients. The complications we examined were: retinopathy, neuropathy, and nephropathy. We used logistic regression models with covariates to estimate odds ratios. We found that the DRB1∗03:01 allele is a protective factor for complications (OR=0.58; p = 0.03), as is the DQA1∗05:01-DQB1∗02:01 haplotype found in linkage disequilibrium with DRB1∗03:01 (OR= 0.59; p = 0.031). The DRB1∗04:01 allele showed no evidence of association (OR=1.13; p = 0.624), although DRB1∗04:01 showed suggestive evidence when the carriers of the protective DRB1∗03:01 were removed from the analysis. The class II DQA1∗03:01-DQB1∗03:02 haplotype was not associated with complications, but the class I allele B∗39:06 (OR=3.27; P = 0.008) suggested a strong positive association with complications. Our results show that in type 1 diabetes patients, specific HLA alleles may be involved in susceptibility to, or protection from, microvascular complications.
[Show abstract][Hide abstract] ABSTRACT: Background:
Autoimmune and non-autoimmune thyroiditis frequently occur in persons with hepatitis C virus (HCV) infection. Treatment with interferon alpha (IFNα) is also associated with significant risk for the development of thyroiditis. To explore HCV-thyroid interactions at a cellular level, we evaluated whether a human thyroid cell line (ML1) could be infected productively with HCV in vitro.
Methods and results:
ML1 cells showed robust surface expression of the major HCV receptor CD81. Using a highly sensitive, strand-specific reverse transcription polymerase chain reaction assay, positive-sense and negative-sense HCV RNA were detected in ML1 cell lysates at days 3, 7, and 14 postinfection with HCV. HCV core protein was expressed at high levels in ML1 supernatants at days 1, 3, 5, 7, and 14 postinfection. The nonstructural protein NS5A was also detected in ML1 cell lysates by Western blotting. HCV entry into ML1 cells was shown to be dependent on the HCV entry factors CD81 and SR-B1/CLA1, while IFNα inhibited HCV replication in ML1 cells in a dose-dependent manner. Supernatants from HCV-infected ML1 cells were able to infect fresh ML1 cells productively, suggesting that infectious virions could be transferred from infected to naïve thyroid cells in vivo. Additionally, HCV infection of ML1 cells led to increased expression of the pro-inflammatory cytokine IL-8.
For the first time, we have demonstrated that HCV can infect human thyroid cells in vitro. These findings strongly suggest that HCV infection of thyrocytes may play a role in the association between chronic HCV infection and thyroid autoimmunity. Furthermore, the thyroid may serve as an extrahepatic reservoir for HCV viral replication, thus contributing to the persistence of viral infection and to the development of thyroid autoimmunity.
No preview · Article · Dec 2012 · Thyroid: official journal of the American Thyroid Association
[Show abstract][Hide abstract] ABSTRACT: Context:Genetic factors play a major role in the etiology of autoimmune thyroid disease (AITD) including Graves' disease (GD) and Hashimoto's thyroiditis (HT). We have previously identified three loci on chromosomes 10q, 12q, and 14q that showed strong linkage with AITD, HT, and GD, respectively.Objectives:The objective of the study was to identify the AITD susceptibility genes at the 10q, 12q, and 14q loci.Design and Participants:Three hundred forty North American Caucasian AITD patients and 183 healthy controls were studied. The 10q, 12q, and 14q loci were fine mapped by genotyping densely spaced single-nucleotide polymorphisms (SNPs) using the Illumina GoldenGate genotyping platform. Case control association analyses were performed using the UNPHASED computer package. Associated SNPs were reanalyzed in a replication set consisting of 238 AITD patients and 276 controls.Results:Fine mapping of the AITD locus, 10q, showed replicated association of the AITD phenotype (both GD and HT) with SNP rs6479778. This SNP was located within the ARID5B gene recently reported to be associated with rheumatoid arthritis and GD in Japanese. Fine mapping of the GD locus, 14q, revealed replicated association of the GD phenotype with two markers, rs12147587 and rs2284720, located within the NRXN3 and TSHR genes, respectively.Conclusions:Fine mapping of three linked loci identified novel susceptibility genes for AITD. The discoveries of new AITD susceptibility genes will engender a new understanding of AITD etiology.
Full-text · Article · Nov 2012 · The Journal of Clinical Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: The CD40 gene, an important immune regulatory gene, is also expressed and functional on nonmyeloid-derived cells, many of which are targets for tissue-specific autoimmune diseases, including β cells in type 1 diabetes, intestinal epithelial cells in Crohn's disease, and thyroid follicular cells in Graves' disease (GD). Whether target tissue CD40 expression plays a role in autoimmune disease etiology has yet to be determined. In this study, we show that target tissue overexpression of CD40 plays a key role in the etiology of autoimmunity. Using a murine model of GD, we demonstrated that thyroidal CD40 overexpression augmented the production of thyroid-specific Abs, resulting in more severe experimental autoimmune GD (EAGD), whereas deletion of thyroidal CD40 suppressed disease. Using transcriptome and immune-pathway analyses, we showed that in both EAGD mouse thyroids and human primary thyrocytes, CD40 mediates this effect by activating downstream cytokines and chemokines, most notably IL-6. To translate these findings into therapy, we blocked IL-6 during EAGD induction in the setting of thyroidal CD40 overexpression and showed decreased levels of thyroid stimulating hormone receptor-stimulating Abs and frequency of disease. We conclude that target tissue overexpression of CD40 plays a key role in the etiology of organ-specific autoimmune disease.
Full-text · Article · Aug 2012 · The Journal of Immunology