The association between endothelial function and autoimmune thyroiditis induced by iodine excess.

BACKGROUND: Iodine excess (IE) intake leads to lymphocyte dysfunction and contributes to autoimmune thyroiditis (AIT). Abnormal thyroid function is associated with adverse cardiovascular events, endothelial dysfunction is often an early pathophysiological feature in most cardiovascular disease. However, the relationship between iodine and the cardiovascular system is currently unclear. Therefore, the aim of this study was to investigate the effects of IE on endothelial function in mouse model. METHODS: A total of 24 NOD.H-2h4 mice were randomly divided into different groups. A sodium iodide (NaI) group supplied with 0.05% NaI water for 8 weeks. Serum levels of tumor necrosis factors α (TNFα), interleukin-6 (IL-6) and C-reactive Protein (CRP), as well as endothelin-1 (ET-1), von Willebrand factor (VWF) and thrombomodulin (THBD) were detected by Elisa. In addition, the mRNA and protein expression of these genes were measured by RT-PCR and Western blotting. RESULTS: Here, we found the urinary iodine concentration (UIC) was higher in the NaI group compared to the control group. Serum levels of ET-1, VWF, and THBD were also significantly lower in the NaI group, however, CRP serum levels are significantly increased. In aorta, the mRNA and protein expression of ET-1, VWF, THBD were downregulated, however, the expression of IL-6, CRP and TNFα mRNA and protein were upregulated in the NaI group. A correlation analysis showed negative correlation between UIC with ET-1, VWF, and THBD, similarly, negative correlation between CRP with THBD was observed. In addition, positive correlations between UIC with CRP. CONCLUSION: Collectively, in the NOD.H-2h4 mice, IE supplementation had a suppressive effect on endothelial function, and this inhibition maybe due to the increase expression of inflammatory cytokines.

In Vivo Inhibition of MicroRNA-326 in a NOD.H-2h4 Mouse Model of Autoimmune Thyroiditis.

Background: Previous studies reported that various miRNAs participate in autoimmune diseases, but the potential regulatory mechanism of miRNAs in autoimmune thyroiditis (AIT) needs further exploration. Objective: This study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2h4 mice. Materials and Methods: Five-week-old NOD.H-2h4 mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4+IL-17a+ cells and CD4+Ets-1+ cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA. Results: In the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group (P = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4+IL-17a+ cells were decreased significantly (P = 0.001), while CD4+Ets-1+ cells were increased significantly in the LV-sponge group (P = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group (P < 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group (P < 0.05). In addition, in LV-sponge groups, CD4+IL-17a+ cells were decreased, while CD4+Ets-1+ cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection. Conclusion: MiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.

Growth arrest-specific protein 6 (Gas6) attenuates inflammatory injury and apoptosis in iodine-induced NOD.H-2h4 mice.

PURPOSE: Growth arrest-specific protein 6 (Gas6) is a vitamin K-dependent protein that plays an important role in the pathogenesis of autoimmune diseases. The purpose of this study was to explore the expression of Gas6 and its effects on autoimmune thyroiditis (AIT). METHOD: A total of 24 male NOD.H-2h4 mice were randomly assigned to three groups: (1) a control group supplied with regular water; (2) a sodium iodide (NaI) group supplied with 0.005% sodium iodide water; and (3) a group treated with recombinant mouse Gas6 (rmGas6) after iodine supplementation (NaI + Gas6 group). The severity of lymphocytic infiltration in the thyroid was measured through histopathology. Serum levels of tumor necrosis factor α (TNF-α), interleukin (IL) 6 and IL-1ß, as well as anti-thyroglobulin antibody (TgAb) titers were measured using an enzyme-linked immunosorbent assay. In addition, the expression of Gas6, Caspase 3, TAM receptors (Axl and MerTK), nuclear factor κB (NF-κB) and I-kappa-B α (IκB-α) were measured by Western blotting. Finally, the proportions of T cells were determined in the splenocytes of NOD.H-2h4 mice by flow cytometry. RESULTS: The mRNA and protein expression of Gas6 was significantly lower in the NaI group compared to the control group. Serum levels of TgAb, TNF-α, IL-6 and IL-1ß were also significantly higher in the NaI group but were dramatically reduced after rmGas6 injection. The prevalence of thyroiditis and the infiltration of lymphocytes were significantly lower in the NaI + Gas6 group compared to the NaI group. The protein expression of cleaved-Caspase 3, phosphorylation of MerTK, and NF-κB and IκB-α in the thyroid gland were significantly reduced after rmGas6 administration. The proportion of Th1, Th2 and Th17 cells in splenocytes were also significantly reduced after rmGas6 treatment, whereas there was a dramatic increase in the proportion of Treg cells. CONCLUSION: Gas6 exerts an anti-inflammatory effect in a mouse model of AIT and may therefore be a potential therapeutic target.

To reflect human autoimmune thyroiditis, thyroid peroxidase (not thyroglobulin) antibodies should be measured in female (not sex-independent) NOD.H2h4 mice.

NOD.H2h4 mice are the most commonly used model for human autoimmune thyroiditis. Because thyroid autoimmunity develops slowly (over months), NOD.H2h4 mice are usually exposed to excess dietary iodide to accelerate and amplify the process. However, unlike the female bias in human thyroid autoimmunity, autoantibodies to thyroglobulin (TgAb) are reported to be similar in male and female NOD.H2h4 . We sought evidence for sexual dimorphism in other parameters in this strain maintained on regular or iodized water. Without iodide, TgAb levels are higher in males than in females, the reverse of human disease. In humans, autoantibodies to thyroid peroxidase (TPOAb) are a better marker of disease than TgAb. In NOD.H2h4 mice TPOAb develop more slowly than TgAb, being detectable at 6 months of age versus 4 months for the latter. Remarkably, unlike TgAb, TPOAb levels are higher in female than male NOD.H2h4 mice on both regular and iodized water. As previously observed, serum T4 levels are similar in both sexes. However, thyroid-stimulating hormone (TSH) levels are significantly higher in males than females with or without iodide exposure. TSH levels correlate with TgAb levels in male NOD.H2h4 mice, suggesting a possible role for TSH in TgAb development. However, there is no correlation between TSH and TPOAb levels, the latter more important than TgAb in human disease. In conclusion, if the goal of an animal model is to closely reflect human disease, TPOAb rather than TgAb should be measured in older female NOD.H2h4 mice, an approach requiring patience and the use of mouse TPO protein.

IL-34 Expression Is Reduced in Hashimoto's Thyroiditis and Associated With Thyrocyte Apoptosis.

Hashimoto's thyroiditis (HT) is a common autoimmune disease accompanied by lymphocyte infiltration and thyroid tissue destruction. IL-34 was first described in 2008, and its involvement in the development of many autoimmune diseases has been recently identified. However, whether IL-34 is a regulatory factor in HT is unclear. Here, we demonstrate that IL-34 is expressed on thyroid follicular epithelial cells and that IL-34 expression is significantly reduced in thyroid tissue in patients with HT and spontaneous autoimmune thyroiditis (SAT) models. Serum IL-34 levels in patients with HT are also significantly reduced. In addition, IL-34 is associated with thyroid autoantibodies in both thyroid tissue and serum. Furthermore, our data show that IL-34 participates in the apoptosis resistance of thyrocytes in HT induced by CSF-1R and may be a potential indicator for evaluating thyrocyte damage.

Iodine excess did not affect the global DNA methylation status and DNA methyltransferase expression in T and B lymphocytes from NOD.H-2h4 and Kunming mice.

Dysregulated DNA methylation in lymphocytes has been linked to various autoimmune disorders. Excessive iodine intake leads to lymphocyte dysfunction and contributes to autoimmune thyroiditis (AIT) flares in humans and animals. However, whether excessive iodine modifies the DNA methylation status in lymphocytes is unknown. Twenty NOD.H-2h4 mice and 20 Kunming mice were randomly divided into high iodine and control groups. We scored lymphatic infiltration in the thyroid by hematoxylin and eosin (H&E) staining and assayed serum thyroglobulin antibody (TgAb) levels by an indirect enzyme-linked immunosorbent assay. CD3+ T cells and CD19+ B cells were separated by flow cytometry. Global DNA methylation levels were examined by absorptiometry. Methylation of long interspersed nucleotide element-1 (LINE-1) repeats was detected with bisulfite sequencing PCR. Expression of DNA methyltransferase (DNMT) 1, DNMT3a and DNMT3b mRNA and protein were determined by real-time PCR and Western blot, respectively. We observed evident thyroiditis in the high­iodine-treated NOD.H-2h4 mice, while mice in the other three groups did not develop thyroiditis. No differences were found in the global methylation levels and methylation status of LINE-1 repeats in T and B lymphocytes from high­iodine-treated NOD.H-2h4 mice and Kunming mice compared with those from normal­iodine-supplemented controls. We did not find obvious changes in DNMT mRNA and protein expression levels in T and B lymphocytes among the studied groups. In conclusion, we showed for the first time that excess iodine did not affect the global methylation status or DNMT expression in T and B lymphocytes in NOD.H-2h4 and Kunming mice.

Glycyrrhizin, a Direct HMGB1 Antagonist, Ameliorates Inflammatory Infiltration in a Model of Autoimmune Thyroiditis via Inhibition of TLR2-HMGB1 Signaling.

BACKGROUND: High mobility group box-1 (HMGB1), a non-histone protein, plays an important role in autoimmune diseases. However, the significance of HMGB1 in the pathogenesis of autoimmune thyroiditis has not been reported. The purpose of this study was to explore whether HMGB1 participates in the pathogenesis of autoimmune thyroiditis, and whether glycyrrhizin (GL), a direct inhibitor of HMGB1, attenuates the severity of thyroid inflammatory infiltration in a murine model of autoimmune thyroiditis. METHODS: A total of 80 male NOD.H-2h4 mice were randomly divided into a control or iodine supplement (NaI) group at four weeks of age, and the control group was fed with regular water, whereas the NaI group was supplied with 0.005% sodium iodine water. Another 24 male NOD.H-2h4 mice were also randomized into three groups (eight mice per group) as follows: control, NaI, and GL treatment after iodine supplementation (NaI + GL). The NOD.H-2h4 mice were fed with 0.005% sodium iodide water for eight weeks to enhance autoimmune thyroiditis. After iodine treatment, the mice received intraperitoneal injections of GL for four weeks. The severity of lymphocytic infiltration in the thyroid gland was measured by histopathological studies. The serum levels of HMGB1, tumor necrosis factor alpha, interleukin (IL)-6, IL-1ß, and thyroglobulin antibody titers were measured using an enzyme-linked immunosorbent assay. HMGB1 expression was measured by immunohistochemical staining and real-time polymerase chain reaction. TLR2, HMGB1, MyD88, and nuclear transcription factor κB were measured by Western blot. RESULTS: The mRNA expression of HMGB1 was significantly higher at 8 and 16 weeks in the NaI group than it was in the control group. Serum levels of thyroglobulin antibodies, HMGB1, tumor necrosis factor alpha, IL-6, and IL-1ß were significantly increased in the NaI group, but they were dramatically attenuated with GL injection. The prevalence of thyroiditis and the infiltration of lymphocytes were significantly decreased in the NaI + GL group. GL administration also significantly reduced the protein expression of TLR2, MyD88, HMGB1 and nuclear transcription factor κB in the thyroid gland and attenuated the severity of thyroiditis. CONCLUSION: HMGB1 may play a crucial role in autoimmune thyroiditis by causing inflammatory infiltration, thus increasing the severity of autoimmune thyroiditis. GL effectively attenuated thyroiditis in the iodine-induced NOD.H-2h4 mice via a molecular mechanism related to the inhibition of TLR2-HMGB1 signaling.

Genes and Environment as Predisposing Factors in Autoimmunity: Acceleration of Spontaneous Thyroiditis by Dietary Iodide in NOD.H2h4 Mice.

In the field of autoimmune thyroiditis, NOD.H2h4 mice have attracted significant and increasing attention since they not only develop spontaneous disease but they present thyroiditis with accelerated incidence and severity if they ingest iodide through their drinking water. This animal model highlights the interplay between genetic and dietary factors in the triggering of autoimmune disease and offers new opportunities to study immunoregulatory parameters influenced by both genes and environment. Here, we review experimental findings with this mouse model of thyroiditis.

Genes and environment as predisposing factors in autoimmunity: acceleration of spontaneous thyroiditis by dietary iodide in NOD.H2(h4) mice.

In the field of autoimmune thyroiditis, NOD.H2(h4) mice have attracted significant and increasing attention since they not only develop spontaneous disease but they present thyroiditis with accelerated incidence and severity if they ingest iodide through their drinking water. This animal model highlights the interplay between genetic and dietary factors in the triggering of autoimmune disease and offers new opportunities to study immunoregulatory parameters influenced by both genes and environment. Here, we review experimental findings with this mouse model of thyroiditis.

Effects of chronic iodine excess on thyroid function and structure in autoimmune-prone NOD.H-2h4 mice / 中华内分泌代谢杂志

To explore the dose- and time- dependent relationship between the chronic iodine excess and thyroid structure, ultrastructure, and thyroid function in autoimmune-prone NOD. H-2h4 mice. Chronic iodine excess leads to iodine-induced goiter with an ultrastructure of follicle epithelial cells injury in a dose and time dependent way.

A study on the mechanism of iodine-induced thyroid epithelial cell injury in the induction of autoimmune thyroiditis / 中华内科杂志

Objective To investigate the effect of iodine excess on thyroid follicle epithelial ultrstructure and the relationship between thyroid iniury and autoimmune thyroiditis. Methods NOD.H-2h4 mice and Kunming mice were randomly divided into four groups receiving plain water,5 fold,10 fold,and 100 fold excessive iodine water.4,8 and 24 weeks after receiving iodine water,the mice were killed.After fixation with osmic acid and dual staining with uranyl chloride and citrate lead,thyroid gland ultrstructure was examined with electron microscopy.Resuits Iodine treated NOD.H-2h4 mice exhibited marked accumulation of peroxisome and secondary lysosomes,apoptosis and necrosis of thyroid epithelial cell.damage of thyroid follicles and lymphocvtic infiltration.The observed changes induced by iodine were in a dose dependent way.Conclusion The oxidative iniury on the thyroid epithelial cells induced by iodine excess might be the prerequisite for the creation of autoimmune thyroiditis.

Experimental investigation on iodine-induced autoimmune thyroiditis in NOD.H-2~(h4) mice / 中华内分泌代谢杂志

Objective To investigate the kinetic changes of inflammatory cell infiltration and thyroid autoantibodies in the development of iodine-induced autoimmune thyroiditis in NOD.H-2~(h4)mice.Methods Either 128 five-week-old NOD.H-2~(h4)mice or 128 Kunming mice were randomly divided into two groups,and received plain water or water containing 0.05% sodium iodide.At the time points of 1,2,4,8,12,16,and 24 week after receiving iodinated water,mice were anesthetized by diethyl ether and bled from eye socket vein,and their thyroid glands were collected.Indirect ELISA method was used to measure the levels of serum thyroglobulin autoantibodies (TgAb)and thyroid hormone.After being fixed with paraformaldehyde and embedded in paraffin,thyroid sections were stained with HE and used for morphometrieal analysis.Results In the iodine treated group of NOD.H-2~(h4) mice,autoimmune thyroiditis was observed as early as 1 week after they began receiving indinated water.The prevalence as well as the degree of autoimmune thyroiditis reached the maximum at 12 weeks and remained until 24 weeks.Serum TgAb level increased after 8 weeks of iodine ingestion in NOD.H-2~(h4) mice,then increased steadily throughout the 24 weeks of experiment.On the contrary,serum TgAb was not increased in the control group of Kunming mice.Conclusion Iodine may induce and exacerbate lymphocytic infiltration of the thyroid in genetically susceptible NOD.H-2~(h4) mice,and serum TgAb is just a marker of autoimmune thyroiditis.