Model For Hashimoto's Thyroiditis Research Articles

A machine learning-based diagnosis modeling of IgG4 Hashimoto's thyroiditis.

This study aims to develop a non-invasive diagnosis model using machine learning (ML) for identifying high-risk IgG4 Hashimoto's thyroiditis (HT) patients. A retrospective cohort of 93 HT patients and a prospective cohort of 179 HT patients were collected. According to the immunohistochemical and pathological results, the patients were divided into IgG4 HT group and non-IgG4 HT group. Serum TgAb IgG4 and TPOAb IgG4 were detected by ELISAs. A logistic regression model, support vector machine (SVM) and random forest (RF) were used to establish a clinical diagnosis model for IgG4 HT. Among these 272 patients, 40 (14.7%) were diagnosed with IgG4 HT. Patients with IgG4 HT were younger than those with non-IgG4 HT (P < 0.05). Serum levels of TgAb IgG4 and TPOAb IgG4 in IgG4 HT group were significantly higher than those in non-IgG4 HT group (P < 0.05). There were no significant differences in gender, disease duration, goiter, preoperative thyroid function status, preoperative TgAb or TPOAb levels, and thyroid ultrasound characteristics between the two groups (all P > 0.05). The accuracy, sensitivity, and specificity were 57%, 78%, and 79% for logistic regression model of IgG4 HT, 80 ± 7%, 84.7% ± 2.6%, and 75.4% ± 9.6% for the RF model and 78 ± 5%, 89.8% ± 5.7%, and 64.7% ± 5.7% for the SVM model. The RF model works better than SVM. The area under the ROC curve of RF ranged 0.87 to 0.92. A clinical diagnosis model for IgG4 HT established by RF model might help the early recognition of the high-risk patients of IgG4 HT.

Puerarin Alleviates Experimental Autoimmune Thyroiditis by Regulating Macrophages.

Hashimoto's thyroiditis (HT) is the most common organ-specific autoimmune disease, predominantly affecting women. Although the pathogenesis of HT is incompletely understood, some studies have found that macrophage polarization plays a role. Puerarin is a soy isoflavone compound that has anti-inflammatory and immunomodulatory effects and regulates macrophage immune activity. This study aimed to verify the therapeutic effect of puerarin on HT and explored its regulatory effect on macrophage polarization imbalance in HT. Through bioinformatics analysis and molecular biology methods, it was found that macrophages increased significantly in HT patients and model mice. Immunological staining showed that puerarin intervention could reduce tissue inflammatory cell infiltration. Molecular biological examination displayed that puerarin could inhibit local and systemic inflammation levels, and the expression of marker thyroglobulin and thyroid peroxidase Abs. In vivo experimental results indicated that puerarin regulated macrophage polarity and reduced inflammatory damage, possibly by inhibiting the pyroptosis signaling pathway. In vivo macrophage clearance experiments demonstrated that puerarin relied on macrophages to exert its mechanism of action in treating HT. The results of this study indicate that macrophages are important mediators in the development of HT, and puerarin can regulate macrophage polarity and inflammatory status to provide thyroid tissue protection, which provides a new idea for the treatment of HT.

Inhibiting Soluble Epoxide Hydrolase Suppresses NF-κB p65 Signaling and Reduces CXCL10 Expression as a Potential Therapeutic Target in Hashimoto's Thyroiditis.

Although Hashimoto's thyroiditis (HT) is one of most common autoimmune thyroid diseases, its treatment remains focused on symptom relief. The soluble epoxide hydrolase (sEH) shows potential functions as a drug target in alleviating some autoimmune diseases; however, we seldom know its role in HT. The protein expression of sEH and related downstream molecules were evaluated by immunohistochemistry, Western blotting, ELISA, or immunofluorescence staining. RNA sequencing of tissue samples was performed to analyze differential genes and dysregulated pathways in HT and controls. The thyroid follicular epithelial cells (TFECs) and rat HT model were used to verify the biological function of sEH and the inhibition role of adamantyl-ureido-dodecanoic acid (AUDA) in HT. The sEH was significantly upregulated in HT patients compared with healthy individuals. Transcriptome sequencing showed cytokine-related pathways and chemokine expression; especially chemokine CXCL10 and its receptor CXCR3 were aberrant in HT patients. In TFECs and a rat HT model, blocking sEH by AUDA inhibitor could effectively inhibit the autoantibody, proinflammatory nuclear kappa factor B (NF-κB) signaling, chemokine CXCL10/CXCR3 expression, and type-1 helper CD4+ T cells. Our findings suggest that sEH/NF-κB p65/CXCL10-CXCR3 might be promising therapeutic targets for HT.

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model.

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT). EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments. SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine. A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Rs7537605 polymorphism in VAV3 gene and rs28665122 polymorphism in SEPS gene are not associated with Hashimoto's thyroiditis in North-East Algerian population.

Hashimoto's thyroiditis (HT) is the most common form of autoimmune thyroid disease which leads, in most cases, to hypothyroidism. HT is also classified as a multifactorial disease, which is caused by an interaction between genetic and environmental factors. Current knowledge of HT genetics is still very limited, especially in Algerian population. We wanted to investigate the association of two single-nucleotide polymorphisms (SNPs) inside VAV3 and SEPS genes with HT in Algerian population. We conducted a case-control study that included 100 HT cases and 126 healthy controls that were recruited from three private endocrinology clinics. Two SNPs, rs7537605 and rs28665122 inside VAV3 and SEPS genes were genotyped using real-time polymerase chain reaction (real-time PCR). Binary logistic regression model was used to test the association of selected SNs with HT and linear regression model was used to test association of these SNPs with thyroid peroxidase antibodies (TPOAb) levels. Binary logistic regression results revealed no allelic association of the minor allele A between Hashimoto's thyroiditis cases and healthy controls (P=0.896) for the rs7537606 in VAV3 gene. The same observation was reported for the AA (P=0.477), AG (P=0.752) genotypes and for the genotypic models: dominant (P=1.0) and recessive (P=0.555). Also, there was no significant difference in the TT (P=0.230), TC (P=0.717) and allelic distribution of the minor allele T (P=0.859), and the combined models: TT + TC (P=1.0), TC + CC (P=0.138) between patients and controls for the rs28665122 polymorphism of the SEPS1 gene. This is the first genetic study that investigated the genetic association of rs7537605 and rs28665122 inside VAV3 and SEPS genes in Algerian population. Our results suggest that these two SNPs may not be involved in the pathogeneses of HT since we found no association between them and HT/TPOAb levels. Further research that will include larger sample size is required.

Hashimoto's Thyroiditis Induces Hippocampus-Dependent Cognitive Alterations by Impairing Astrocytes in Euthyroid Mice.

Background: Although studies have reported an increased risk for cognitive disorders in Hashimoto's thyroiditis (HT) patients, even in the euthyroid state, the mechanisms involved remain unclear. The hippocampus is a classic brain region associated with cognitive function, among which the formation of long-term potentiation (LTP) in the Schaffer collateral-CA1 pathway plays an important role in the process of learning and memory. Therefore, this study established a euthyroid HT model in mice and investigated whether and how HT itself has the ability to trigger LTP alterations accompanied by learning and memory abnormality. Methods: An experimental euthyroid HT model was established in NOD mice through immunization with porcine thyroglobulin (Tg). Morris water maze was measured to determine mice spatial learning and memory. We investigated the effect of HT on synaptic transmission and high-frequency stimulation-induced LTP in the Schaffer collateral-CA1 synapse of mice hippocampus in vivo. Then, animals were sacrificed for thyroid-related parameter measure as well as detection of cellular and molecular events associated with the induction of LTP. Results: HT mice showed intrathyroidal lymphocyte infiltration and rising serum thyroid autoantibody levels accompanied by normal thyroid function. The HT mice had poorer performance in Morris water maze than controls. These alterations were mirrored by abnormalities in synaptic plasticity in the Schaffer collateral-CA1 synapses of the hippocampus in vivo. The integrity of the synaptic structure is the premise for the production of LTP. As detected by transmission electron microscopy, the ultrastructure of synapse and astrocyte in the hippocampus were impaired in euthyroid HT mice. Additionally, Western blot and real-time polymerase chain reaction analyses confirmed that in HT mice, GS, GLAST, and GLT-1, key elements in glutamate-glutamine circulation located in astrocyte, were downregulated, accompanied by elevated levels of glutamate in the hippocampus, which impaired the material basis for LTP induction. NMDR2B expression in the hippocampus was also downregulated. Conclusion: HT can induce damage of LTP in the hippocampal Schaffer collateral-CA1 pathway in the euthyroid state, and this can be attributed, at least partly, to astrocytes impairment, which may underlie the deleterious effects of HT itself on hippocampal-dependent learning and memory function.

Role of Nampt overexpression in a rat model of Hashimoto's thyroiditis and its mechanism of action.

The present study was designed to investigate the role of nicotinamide phosphoribosyltransferase (Nampt) overexpression in a rat model of Hashimoto's thyroiditis (HT) and its mechanism of action. A rat model of HT was constructed, and the HT rats were injected with an adenoviral expression vector carrying the Nampt gene. The expression of Nampt and Toll-like receptor 4 (TLR4) in thyroid tissues was examined using immunohistochemistry (IHC), RT-qPCR and western blot analyses. Serum anti-thyroglobulin antibodies (TGAb) and anti-thyroid peroxidase antibodies (TPOAb) were measured using chemiluminescence method. Hematoxylin and eosin (H&E) and IHC staining of the rat thyroid tissues showed destroyed thyroid follicles and monocyte infiltration, as well as increased Nampt expression in the thyroid tissues of rats with HT. Furthermore, it was found that Nampt overexpression led to increased severity of inflammatory infiltration in thyroid tissues and increased levels of TPOAb in the serum of HT rats; however, the serum TGAb level was not affected by Nampt overexpression. In addition, Nampt overexpression promoted TLR4 expression in HT rats. In conclusion, it was demonstrated that Nampt was strongly expressed in the capillary region of HT rats thyroid tissues. The Nampt mRNA level was increased but the Nampt protein level was decreased in the thyroid tissues of rats with HT. Nampt overexpression has a promotive effect on HT progression, and this effect was related to TLR4. This study suggests that inhibition of Nampt activity may be valuable in the treatment of HT.

Depletion of Regulatory T Cells in Visceral Adipose Tissues Contributes to Insulin Resistance in Hashimoto's Thyroiditis.

Hashimoto's Thyroiditis (HT) is a common organ-specific autoimmune disorder associated with a high incidence, and insulin resistance is highly related to autoimmune. Here, we examined the insulin sensitivity in HT patients and found decreased insulin sensitivity occurred in HT patients. To explore the relationship between impaired insulin sensitivity and immune status, we established HT model mice which showed similar pathological features and immune features to HT patients. In HT model mice, reinfusion of regulatory T cells (Tregs) from peripheral blood of normal mice could improve insulin sensitivity and decrease the inflammation. Anti-CD25 antibodies blocked beneficial effects from reinfusion of Tregs, but delayed administration of anti-CD25 antibodies could not abolished the effect from Tregs. Delayed administration of anti-CD25 antibodies abolished exogenous Tregs in peripheral blood, but there were increased exogenous Tregs located to visceral adipose tissues (VATs) which modulated the expression of cytokines in VATs. These findings suggest that insulin resistance exists in HT patients and it associates with the decreased Tregs and increased inflammation in the VATs.

Molecular amino acid signatures in the MHC class II peptide-binding pocket predispose to autoimmune thyroiditis in humans and in mice

Hashimoto's thyroiditis (HT) is associated with HLA, but the associated allele is still controversial. We hypothesized that specific HLA-DR pocket-sequence variants are associated with HT and that similar variants in the murine I-E locus (homologous to HLA-DR) predispose to experimental autoimmune thyroiditis (EAT), a classical mouse model of HT. Therefore, we sequenced the polymorphic exon 2 of the HLA-DR gene in 94 HT patients and 149 controls. In addition, we sequenced exon 2 of the I-E gene in 22 strains of mice, 12 susceptible to EAT and 10 resistant. Using logistic regression analysis, we identified a pocket amino acid signature, Tyr-26, Tyr-30, Gln-70, Lys-71, strongly associated with HT (P = 6.18 x 10(-5), OR = 3.73). Lys-71 showed the strongest association (P = 1.7 x 10(-8), OR = 2.98). This association was seen across HLA-DR types. The 5-aa haplotype Tyr-26, Tyr-30, Gln-70, Lys-71, Arg-74 also was associated with HT (P = 3.66 x 10(-4)). In mice, the I-E pocket amino acids Val-28, Phe-86, and Asn-88 were strongly associated with EAT. Structural modeling studies demonstrated that pocket P4 was critical for the development of HT, and pockets P1 and P4 influenced susceptibility to EAT. Surprisingly, the structures of the HT- and EAT-susceptible pockets were different. We conclude that specific MHC II pocket amino acid signatures determine susceptibility to HT and EAT by causing structural changes in peptide-binding pockets that may influence peptide binding, selectivity, and presentation. Because the HT- and EAT-associated pockets are structurally different, it is likely that distinct antigenic peptides are associated with HT and EAT.

CD4 +CD25 + naturally occurring regulatory T cells and not lymphopenia play a role in the pathogenesis of iodide-induced autoimmune thyroiditis in NOD-H2 h4 mice

NOD-H2 h4 mice, which express I-A k on the NOD background, spontaneously develop autoimmune thyroiditis, a model of Hashimoto thyroiditis in humans, by adding iodide in the drinking water. Parental NOD mice have previously been shown to have intrinsic numerical abnormalities in peripheral lymphocytes and lymphocyte subpopulations such as CD4 +CD25 + naturally occurring regulatory T cells (Treg). Therefore we first investigated whether the similar abnormalities exist in NOD-H2 h4 mice. We observed that, compared with other non-autoimmune disease prone BALB/c and C57BL/6 mice, NOD-H2 h4 mice have lower numbers of splenocytes, CD3 +T, CD4 +T and CD8 +T cells but the ratios of Treg to CD4 +T cells were comparable. Increasing the numbers of peripheral lymphocytes by Complete Freund's Adjuvant immunization or splenocyte transfer did not affect development of thyroiditis, indicating that lymphopenia does not play a critical role in the pathogenesis of thyroiditis. We next examined the significance of Treg by depleting this lymphocyte subset with anti-CD25 antibody. Treg depletion, performed 4 days before the administration of NaI water for 8 weeks, significantly exacerbated thyroiditis ( p < 0.01). Anti-thyroglobulin antibody titers also increased by Treg depletion ( p < 0.01) without changing the IgG2b to IgG1 ratios. In addition, expression levels of mRNA for IFN-γ and IL-4 were enhanced in parallel. However, T 4 levels were similar between antibody-treated and untreated groups. Additional anti-CD25 administration at 3 weekly intervals did not influence these results. These data, together with previous studies on other mouse models of inducible thyroiditis and Graves’ disease, indicate the role played by Treg in keeping anti-thyroid autoimmune reaction in check in experimental autoimmune thyroid diseases.

Graves' hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA.

Familial and twin studies in Caucasians have established that the MHC class II allele HLA-DRB1*0301 (DR3) is a strong susceptibility gene in Graves' hyperthyroid disease (GD). To determine if a DR3 transgene could help establish an animal model for GD, we expressed DR3 molecules in class II-knockout NOD mice (H2Ag7-). DR3+g7- mice were given cardiotoxin prior to immunization on weeks 0, 3 and 6 with plasmid DNA encoding human thyrotropin receptor (TSHR). Two groups of mice were also coimmunized with plasmid DNA for IL-4 or GM-CSF. Serial bleeds on weeks 8, 11 and 14 showed that approximately 20% of mice produced thyroid-stimulating antibodies (Abs), and approximately 25% had elevated T4 levels. In particular, a subset displayed both signs of hyperthyroidism, resulting in approximately 30% with some aspect of GD syndrome. Additional mice had thyroid-stimulating blocking Abs and/or TSH-binding inhibitory immunoglobulins, while most mice showed strong labelling of TSHR+ cells by flow cytometry. Interestingly, lymphocytic infiltration with thyroid damage and Abs to mouse thyroglobulin were also noted. Vector controls were uniformly negative. Thus, DR3 transgenic mice can serve as a model for GD, similar to our earlier reports that this allele is permissive for the Hashimoto's thyroiditis model induced with human thyroglobulin.

Fas and Fas ligand gene expression in autoimmune thyroiditis in BB/W rats.

Apoptosis via the Fas pathway is a potential mechanism for thyroid tissue destruction leading to clinical hypothyroidism in Hashimoto's thyroiditis (HT). Recent studies reported contradictory results regarding the regulation of Fas/Fas ligand (FasL) expression by cytokines in vitro. We therefore determined the Fas and FasL gene expression in the BioBreeding/Worcester (BB/W) rat thyroiditis model, which can be regarded as a model for HT. In order to obtain BB/W rats with spontaneous, iodine-induced or without lymphocytic thyroiditis (LT), rats were divided into 3 groups: 55-day-old rats after 24 days of iodine administration, 75-day-old rats after 45 days of iodine administration, and 101-day-old rats respectively. The gene expression of Fas, FasL, and interleukin (IL)-1beta was determined by Genescan fragment analysis using reverse polymerase chain reaction. Serum thyroglobulin (TG) antibody concentrations were measured and the extent of lymphocytic infiltration of one thyroid lobe was histologically graded. Fas and FasL gene expression was significantly higher in rats with LT and correlated with the extent of lymphocytic infiltration and the TG antibody level. There was no evidence that the expression of IL-1beta or other cytokines is related to the expression of Fas or its ligand. The increased expression of Fas and FasL in LT of BB/W rats suggests the involvement of the Fas pathway in the pathogenesis of LT in BB/W rats. However, in contrast to results of recent in vitro studies, in the BB/W rat Fas/FasL expression is not regulated by IL-2, -4, -6, -10, -12, interferon gamma, and tumor necrosis factor alpha.

Cloning and Characterization of Murine Thyroglobulin cDNA

Thyroglobulin is used to induce in mice experimental autoimmune thyroiditis (EAT), a model for Hashimoto thyroiditis. Because murine thyroglobulin is a more potent inducer of EAT than heterologous thyroglobulins, it has been hypothesized that it contains unique pathogenic epitopes. The validation of this hypothesis has been hampered by the lack of the murine thyroglobulin sequence. To identify murine-specific areas in thyroglobulin, we cloned, by reverse transcriptase PCR, and sequenced the complete murine thyroglobulin cDNA. This encodes a polypeptide of 2748 amino acids that is 73.5 and 71.8% identical to bovine and human thyroglobulin, respectively. Six regions are unique to each species. We also analyzed through EpiMer the sequences able to bind to the I-Ekmajor histocompatibility allele and, therefore, function as T cell epitopes. EpiMer analysis showed seven murine-specific T cell epitopes in thyroglobulin. The availability of the complete murine thyroglobulin sequence should promote the understanding of the pathogenesis and immunoregulation of EAT.

HLA-DRB1 polymorphism determines susceptibility to autoimmune thyroiditis in transgenic mice: definitive association with HLA-DRB1*0301 (DR3) gene.

Familial clustering of autoimmune thyroid diseases has led to studies of their association with human major histocompatibility complex (MHC) class II genes. One such gene implicated in Hashimoto's thyroiditis (HT) is HLA-DR3, but the association is weak and is contradicted by other reports. On the other hand, murine experimental autoimmune thyroiditis (EAT), a model for HT, presents a clear linkage with MHC class II. Moreover, it is inducible with thyroglobulin (Tg), the common autoantigen in either species. Immunization of HLA-DRB1* 0301 (DR3) transgenic mice with mouse or human Tg resulted in severe thyroiditis. In contrast, transgenic mice expressing the HLA-DRB1*1502 (DR2) gene were resistant to EAT. Our studies show that HLA-DRB1 polymorphism determines susceptibility to autoimmune thyroiditis and implicate Tg as an important autoantigen.