Development Of Experimental Autoimmune Thyroiditis Research Articles

Content of macro- and microelements in rats with experimental autoimmune thyroiditis and under the influence of lemna minor

The aim was to study the effect of aqueous extract and 30 % alcohol tincture of Lemna minor frond (AELM and TLM) on the content of macro- and microelements in rats with experimental autoimmune thyroiditis. Materials and methods. The effect of AELM and TLM was studied in a model of experimental autoimmune thyroiditis in rats, induced by immunization with human thyroid antigen. The serum levels of total thyroxine, total triiodothyronine, antibodies to thyroglobulin, antibodies to thyroperoxidase, and concentrations of macro- and microelements were studied. Results. The development of experimental autoimmune thyroiditis led to a decrease in the content of total thyroxine and antibodies to thyroperoxidase in the blood serum, an increase in the content of antibodies to thyroglobulin and a decrease in the concentrations of sodium, chlorine, potassium phosphorus, zinc, copper, iron and magnesium. It was found that the use of AELM and TLM against the background of autoimmune thyroiditis led to the restoration of T4 and elemental balance in rats, which was manifested in an increase in the content of sodium, chlorine, phosphorus, magnesium, zinc, iron, copper and calcium in the blood serum. It was also shown that TLM had a more powerful effect on the normalisation of the content of such elements as chlorine, potassium, copper and zinc. The obtained effect of AELM and TLM on the content of macro- and microelements in the blood serum of animals with experimental AIT can be explained by their positive effect on the functional activity of the thyroid gland. The studied extract and tincture may also affect other physiological and biochemical processes due to the content of macro- and microelements and other biologically active substances, which requires further research. Conclusions. Administration of AELM and TLM to rats with experimental AIT contributed to lowering the level of Anti-TG and restoring the thyroid and elemental status in the animals' blood serum. The obtained research data allow us to recommend AELM and TLM as a regulator of the elemental status of the body in case of changes in thyroid function

Notch Signaling Pathway Promotes Th17 Cell Differentiation and Participates in Thyroid Autoimmune Injury in Experimental Autoimmune Thyroiditis Mice.

To investigate whether the Notch signaling pathway participates in the occurrence and development of experimental autoimmune thyroiditis (EAT) by affecting the differentiation and function of Th17 cells. Experimental mice were randomly divided into a control group, an EAT-A group (porcine thyroid immunoglobulin- (pTg-) treated mice) and an EAT-B group (treated with the DAPT γ-secretase inhibitor before pTg). HE staining, IHC staining, flow cytometry, RT-qPCR, and ELISA were used to evaluate the degrees of thyroiditis, detect the percentage of Th17 cells and measure the expression of retinoic acid-related orphan receptor gamma t (RORγt), interleukin-17A (IL-17A), and the main components of the Notch signaling pathway. The degrees of thyroiditis, the proportions of Th17 cells, and the expression of RORγt and IL-17A were significantly decreased in the EAT-B group after blocking the Notch signaling pathway by DAPT, and these parameters were significantly increased in the EAT-A group compared to the control group (all P < 0.05). Additionally, the Th17 cell percentages and IL-17A concentrations in spleen mononuclear cells (SMCs) from EAT-A mice decreased in a dose-dependent manner after DAPT treatment in vitro (all P < 0.01). Correlation analyses revealed that the Th17 cell percentages were positively correlated with the serum TgAb titers, Notch pathway-related mRNA expression levels, and IL-17A concentrations in EAT mice (all P < 0.05). The expression of Notch signaling pathway components was upregulated in EAT mice, but blockade of the Notch signaling pathway alleviated the degree of thyroiditis, decreased the Th17 cell proportions, and downregulated the IL-17A effector cytokine both in vivo and in vitro. These findings suggested that the Notch signaling pathway may be involved in the pathogenesis of thyroid autoimmune injury in EAT mice by promoting the differentiation of Th17 cells.

Estrogen receptor β activation stimulates the development of experimental autoimmune thyroiditis through up-regulation of Th17-type responses

Estrogens play important roles in autoimmune thyroiditis, but it remains unknown which estrogen receptor (ER) subtype mediates the stimulatory effects. Herein we treated ovariectomized mice with ERα or ERβ selective agonist followed by thyroglobulin-immunization to induce experimental autoimmune thyroiditis (EAT), and observed the aggravation of EAT after diarylpropionitrile (DPN, ERβ selective agonist) administration. The mRNA levels of interleukin(IL)-17A, IL-21 and RORγt and percentages of T helper (Th) 17 cells were up-regulated in the splenocytes of DPN-treated mice. Activated ERβ was found directly binding to IL-17A and IL-21 gene promoters, and also indirectly promoting IL-21 and RORγt gene transcription through interaction with NF-κB. The expressions of co-stimulatory molecules were increased on antigen-presenting cells (APCs) after DPN administration. It suggests that ERβ is the predominant ER subtype responsible for EAT development, and its activation may enhance Th17-type responses through genomic pathways and alteration of APCs' activities.

Physiological low-dose oestrogen promotes the development of experimental autoimmune thyroiditis through the up-regulation of Th1/Th17 responses

Previous studies have reported a preponderance of autoimmune thyroiditis (AIT) in females, but the detailed mechanisms have not been elucidated. In this study, we explored the effects of oestrogen on experimental AIT (EAT) and its potential mechanisms in an ovariectomised mouse model through the supplementation of high (equivalent to the level during pregnancy) or low (equivalent to the level at the oestrus stage) doses of oestradiol (E2). We found that EAT incidence, the intrathyroidal inflammatory score, serum anti-thyroglobulin IgG2b levels, splenic mRNA expression of Th1- and Th17-specific transcription factors and typical cytokines and the proportion of IL-12-producing dendritic cells were significantly increased in EAT mice treated with low-dose E2 compared with those in the control group. However, they were not changed when administered with high-dose E2. These findings indicate that low physiological levels of E2 can stimulate the occurrence and development of EAT through the up-regulation of Th1/Th17 responses.

OX40L/Jagged1 Cosignaling by GM-CSF–Induced Bone Marrow-Derived Dendritic Cells Is Required for the Expansion of Functional Regulatory T Cells

Abstract Earlier, we had demonstrated that treatment with low dose of GM-CSF can prevent the development of experimental autoimmune thyroiditis (EAT), experimental autoimmune myasthenia gravis, and type 1 diabetes, and could also reverse ongoing EAT and experimental autoimmune myasthenia gravis. The protective effect was mediated through the induction of tolerogenic CD11C+CD8α− dendritic cells (DCs) and consequent expansion of Foxp3+ regulatory T cells (Tregs). Subsequently, we showed that GM-CSF acted specifically on bone marrow precursors and facilitated their differentiation into tolerogenic dendritic cells (DCs; GM-CSF–induced bone marrow-derived DCs [GM-BMDCs]), which directed Treg expansion in a contact-dependent manner. This novel mechanism of Treg expansion was independent of TCR-mediated signaling but required exogenous IL-2 and cosignaling from DC-bound OX40L. In this study, we observed that OX40L-mediated signaling by GM-BMDCs, although necessary, was not sufficient for Treg expansion and required signaling by Jagged1. Concurrent signaling induced by OX40L and Jagged1 via OX40 and Notch3 receptors expressed on Tregs was essential for the Treg expansion with sustained FoxP3 expression. Adoptive transfer of only OX40L+Jagged1+ BMDCs led to Treg expansion, increased production of IL-4 and IL-10, and suppression of EAT in the recipient mice. These results showed a critical role for OX40L- and Jagged1-induced cosignaling in GM-BMDC–induced Treg expansion.

GM-CSF-induced CD11c+CD8a--dendritic cells facilitate Foxp3+ and IL-10+ regulatory T cell expansion resulting in suppression of autoimmune thyroiditis

GM-CSF plays an essential role in the differentiation of dendritic cells (DCs). Our studies have shown that GM-CSF treatment can induce semi-mature DCs and CD4+CD25+ regulatory T cells (Tregs) and suppress ongoing autoimmunity in mouse models. In this study, we examined the differences in the potential of GM-CSF to exert tolerogenic function on CD8a+ and CD8a- sub-populations of DCs in vivo. We show that GM-CSF modulates CD8a-, but not CD8a+ DCs in vivo, by inhibiting the surface expression of activation markers MHC II and CD80 and production of inflammatory cytokines such as IL-12 and IL-1beta. Self-antigen [mouse thyroglobulin (mTg)] presentation by GM-CSF-exposed CD8a- DCs to T cells from mTg-primed mice induced a profound increase in the frequency of forkhead box P3 (FoxP3)-expressing T cells compared with antigen presentation by GM-CSF-exposed CD8a+ DCs and control CD8a+ and CD8a- DCs. This tolerogenic property of GM-CD8a- DCs was abrogated when IL-12 was added. GM-CSF-exposed CD8a- DCs could also induce secretion of significantly higher amounts of IL-10 by T cells from mTg-primed mice. Importantly, adoptive transfer of CD8a- DCs from GM-CSF-treated SCID mice, but not untreated mice, into wild-type CBA/J mice prevented the development of experimental autoimmune thyroiditis (EAT) in the recipient animals upon immunization with mTg. Collectively, our results show that GM-CSF renders CD8a- DCs tolerogenic, and these DCs induce Foxp3+ and IL-10+ Tregs.

The “A, B and C” of Her-2 DNA vaccine development

The development of Her-2 DNA vaccine has progressed through three phases that can be categorized as phase "A": the pursuit of Her-2 as a tumor-associated "antigen", phase "B": tilting the "balance" between tumor immunity and autoimmunity and phase "C": the on-going "clinical trials". In phase "A", a panel of human ErbB-2 or Her-2 plasmids were constructed to encode non-transforming Her-2 derivatives. The immunogenicity and anti-tumor activity of Her-2 DNA vaccines were tested in human Her-2 transgenic mice with or without the depletion of regulatory T cells (Tregs). However, Treg depletion or other immune modulating regimens may increase the risk of autoimmunity. In phase "B", the balance between tumor immunity and autoimmunity was assessed by monitoring the development of experimental autoimmune thyroiditis (EAT). To test the efficacy of Her-2 DNA vaccines in cancer patients, clinical trials have been initiated in phase "C". Significant anti-Her-2 and anti-tumor activity was observed when Her-2 transgenic mice were electro-vaccinated after Treg depletion. Susceptibility to EAT was also enhanced by Treg depletion and there was mutual amplification between Her-2 immunity and EAT development. Although Tregs regulate both EAT and Her-2 immunity, their effector mechanisms may differ. It may be possible to amplify tumor immunity with improved strategies that can by-pass undue autoimmunity. Critical information will be revealed in the next decade to expedite the development of cancer vaccines.

Maturation of dendritic cells by necrotic thyrocytes facilitates induction of experimental autoimmune thyroiditis

Dendritic cell (DC) maturation is required for efficient presentation of autoantigens leading to autoimmunity. In this report, we have examined whether release of tissue antigens from necrotic thyroid epithelial cells can trigger DC maturation and initiation of a primary anti-self response. DC were cocultured with either viable (VT/DC) or necrotic (NT/DC) thyrocytes, and their phenotypic and functional maturation as well as immunopathogenic potential were assessed. Significant up-regulation of surface MHC class II and costimulatory molecule expression was observed in NT/DC but not in VT/DC. This was correlated with a functional maturation of NT/DC, determined by IL-12 secretion. Challenge of CBA/J mice with NT/DC, but not with VT/DC, elicited thyroglobulin (Tg)-specific IgG as well as Tg-specific CD4(+) T-cell responses and led to development of experimental autoimmune thyroiditis. These results support the view that thyroid epithelial cell necrosis may cause autoimmune thyroiditis via maturation of intrathyroidal DC.

Targeted engagement of CTLA-4 prevents autoimmune thyroiditis.

The CTLA-4-mediated signal is a critical step in the down-modulation of immune responses. The therapeutic potential of this signal to induce tissue-specific tolerance was investigated by using an anti-CTLA-4 antibody that was coupled to an antibody specific for the thyrotropin receptor. After in vivo administration, this bispecific antibody (BiAb) accumulated in the thyroid and prevented development of experimental autoimmune thyroiditis (EAT) in mice immunized with mouse thyroglobulin (mTg). Lymphocytes from BiAb-treated mice showed a significant reduction in their ability to proliferate, and to produce IL-2, IFN-gamma and tumor necrosis factor (TNF)-alpha, in response to mTg re-stimulation compared to lymphocytes from untreated mice. Moreover, BiAb-treated mice showed suppressed anti-mTg antibody response, lymphocytic infiltration of the thyroid and follicular destruction. The BiAb targeted to the thyroid most likely facilitated engagement of CTLA-4, resulting in an increase in the number of CD4(+)CD25(+) T cells. These regulatory T cells suppressed in vitro mTg-specific T cell responses, which were associated with an enhanced transforming growth factor (TGF)-beta1 production. Neutralization of TGF-beta1 increased mTg-specific in vitro proliferation of, and IL-2 production by, T cells from BiAb-treated mice. Our data suggest that engagement of CTLA-4 expressed on activated autoreactive T cells in close proximity to the thyroid can increase the number of regulatory T cells and their ability to produce TGF-beta1, with a concomitant reduction in IFN-gamma and TNF-alpha, resulting in suppression of EAT.

Enhanced iodination of thyroglobulin facilitates processing and presentation of a cryptic pathogenic peptide.

Increased iodine intake has been associated with the development of experimental autoimmune thyroiditis (EAT), but the biological basis for this association remains poorly understood. One hypothesis has been that enhanced incorporation of iodine in thyroglobulin (Tg) promotes the generation of pathogenic T cell determinants. In this study we sought to test this by using the pathogenic nondominant A(s)-binding Tg peptides p2495 and p2694 as model Ags. SJL mice challenged with highly iodinated Tg (I-Tg) developed EAT of higher severity than Tg-primed controls, and lymph node cells (LNC) from I-Tg-primed hosts showed a higher proliferation in response to I-Tg in vitro than Tg-primed LNC reacting to Tg. Interestingly, I-Tg-primed LNC proliferated strongly in vitro against p2495, but not p2694, indicating efficient and selective priming with p2495 following processing of I-Tg in vivo. Tg-primed LNC did not respond to either peptide. Similarly, the p2495-specific, IL-2-secreting T cell hybridoma clone 5E8 was activated when I-Tg-pulsed, but not Tg-pulsed, splenocytes were used as APC, whereas the p2694-specific T cell hybridoma clone 6E10 remained unresponsive to splenic APC pulsed with Tg or I-Tg. The selective in vitro generation of p2495 was observed in macrophages or dendritic cells, but not in B cells, suggesting differential processing of I-Tg among various APC. These data demonstrate that enhanced iodination of Tg facilitates the selective processing and presentation of a cryptic pathogenic peptide in vivo or in vitro and suggest a mechanism that can at least in part account for the association of high iodine intake and the development of EAT.

Vβ8.2 Transgene Expression Interferes with Development of Experimental Autoimmune Thyroiditis in CBA k/q but Not k/k Mice

The thyroiditogenic T cell receptor (TCR) repertoire is not yet well defined in murine experimental autoimmune thyroiditis (EAT). Our recent work has shown that, while Vβ8+T cells have no major role in EAT induction with mouse thyroglobulin (MTg), Vβ13 may be involved. To examine the effect of skewing the TCR repertoire on EAT development, CBA (H2k) mice were mated with B10.Q mice harboring an ovalbumin-specific Vβ8.2 TCR transgene (trg), and the trg+mice were backcrossed to CBA. FACS analysis showed that peripheral blood T cells from trg+mice had about 76 and 90% Vβ8.2+cells in the CD4+and CD8+subsets, respectively, compared with about 15 and 11% in trg−sibs. The transgenic CBA k/k and k/q mice were immunized with MTg and sacrificed 28 days later. In all trg+mice, anti-MTg titers and T cell proliferative responses to MTg were significantly lowered. However, thyroid infiltration was distinctly different in the two strains of transgenic mice; a significant decrease was seen primarily in k/q, but not k/k, trg+mice. Thus, skewing the TCR repertoire to overexpress an irrelevant TCR revealed the possession of a less flexible thyroiditogenic TCR repertoire in k/q, but not k/k, mice.

Suppression of development of experimental autoimmune thyroiditis by oral administration of thyroglobulin

Experimental autoimmune thyroiditis (EAT), which to some extent represents an experimental model of human chronic lymphocytic thyroiditis, is an organ-specific autoimmune disease characterized by autoantibody production to thyroid antigens (Ag) and mononuclear infiltration of the thyroid gland. EAT induced by immunization with human thyroglobulin (hTG) with Freund's adjuvant in CBA/J (H-2K) mice is associated with prominent B and T cell responses. We report that oral administration of hTG effectively reduces the immune responses in EAT in mice in an Ag-specific manner. Both cellular and humoral immune responses are reduced in a dose-dependent manner. Histological evidence of disease is dramatically reduced. Suppression of the immune responses is seen 2 weeks after Ag challenge, with partial inhibition of proliferative and antibody responses. Six weeks after immunization, further inhibition is observed of both T and B cell responses. Hyporesponsiveness of T and B cell reactivity is seen only to hTG; T and B cell responses to other immunogens are not affected, including purified protein derivative and the nonrelated Ag BSA. This model may provide the basis for immunotherapy of autoimmune thyroid diseases in man.

Suppression of development of experimental autoimmune thyroiditis by oral administration of thyroglobulin.

Experimental autoimmune thyroiditis (EAT), which to some extent represents an experimental model of human chronic lymphocytic thyroiditis, is an organ-specific autoimmune disease characterized by autoantibody production to thyroid antigens (Ag) and mononuclear infiltration of the thyroid gland. EAT induced by immunization with human thyroglobulin (hTG) with Freund's adjuvant in CBA/J (H-2K) mice is associated with prominent B and T cell responses. We report that oral administration of hTG effectively reduces the immune responses in EAT in mice in an Ag-specific manner. Both cellular and humoral immune responses are reduced in a dose-dependent manner. Histological evidence of disease is dramatically reduced. Suppression of the immune responses is seen 2 weeks after Ag challenge, with partial inhibition of proliferative and antibody responses. Six weeks after immunization, further inhibition is observed of both T and B cell responses. Hyporesponsiveness of T and B cell reactivity is seen only to hTG; T and B cell responses to other immunogens are not affected, including purified protein derivative and the nonrelated Ag BSA. This model may provide the basis for immunotherapy of autoimmune thyroid diseases in man.

Protection from experimental autoimmune thyroiditis in CBA mice with the novel immunosuppressant deoxyspergualin.

The effects of the novel immunosuppressant Deoxyspergualin (DSP) on the development of experimental autoimmune thyroiditis (EAT) in CBA mice were studied. For EAT induction, the mice were immunized with 100 micrograms of porcine thyroglobulin (p Tg) emulsified in CFA on day 0 and in IFA, for boosting, on day 14. Twenty-eight days after primary immunization, histological and serological signs of EAT occurred in control mice treated with PBS which showed marked lymphoid infiltration of the thyroid glands along with increased serum titres of anti-pTg antibodies. Development of both these EAT features was significantly suppressed when the mice were treated with 2.5 mg/kg body weight DSP, given daily, five times a week, from day -2 to day +28 after immunization. The effect appeared to be dose-dependent and DSP was ineffective when given under the same experimental conditions at the dose of 0.5 mg/kg body weight. No DSP-toxic effects could be observed during the experiment. These results provide further evidence for the powerful immunosuppressive properties of DSP and suggest that this drug may be used in the treatment of autoimmune thyroid diseases and other T-cell mediated autoimmune disorders in humans.

Role of Late Complement Components in Experimental Autoimmune Thyroiditis

Availability of a line of rabbits deficient in the sixth complement component (C6-D) made it possible to evaluate the role of the terminal complement complex (TCC) in the development of experimental autoimmune thyroiditis (EAT) of the rabbit. Immunization with saline extract of homologous thyroid, known to be composed predominantly of thyroglobulin, led in normocomplementemic (NC) rabbits to severe thyroidiris, with cellular infiltrates occupying 50-95% of the thyroid, and to minimal or moderate thyroidiris, with 1-35% of thyroids infiltrated in C6-D rabbits. Cellular infiltrates consisted predominantly of mononuclear cells with appreciable numbers of granulocytes. Destruction of thyroid follicles was extensive and diffuse in NC rabbits, but it was only minimal and focal in C6-D rabbits. Immunohistology revealed in both groups of rabbits deposits of IgG and C3 along follicular basal laminae. In addition, NC rabbits showed deposition of C6 and MAC in thyroid follicles. These results suggested that TCC is necessary for the development of fully expressed, severe EAT; simultaneously, however, they showed that a significantly reduced EAT can develop without TCC. Administration of NC but not of C6-D rabbit serum to C6-D rabbits resulted in a significant increase in the severity of EAT. It was also shown that C6-D rabbits have "normal" T-cell activity, since they developed experimental autoallergic encephalomyelitis as readily as NC rabbits. Therefore, it is likely that development of EAT is indeed impaired by the C6 deficiency in rabbits. The requirement for TCC observed in this study may be relevant to the understanding of the pathogenesis of Hashimoto's thyroiditis, in which thyroid tissue was recently shown to contain TCC deposits.

Tissue specific binding of lymphocytes to the thyroid gland of BB/W rats may be an early event in the development of thyroiditis.

Thyroiditis occurs in about 50% of diabetic Bio Breeding/Worcester (BB/W) rats. In order to investigate the earliest stages of lymphocyte homing to the thyroid gland in the development of experimental autoimmune thyroiditis, we measured the amount of trapping of peripheral blood lymphocytes (PBL) from BB/W rats to the thyroid gland of syngeneic recipient animals. PBL, from donor normal or diabetic BB/W rats, labelled with 51Cr, were injected, i.v., into normal, "potentially diabetic" or diabetic BB/W recipients. After 24 hr the rats were sacrificed and the radioactivity of selected individual organs counted. Results were calculated as % binding of 51Cr-labelled PBL/unit weight of tissue and expressed as a binding index by comparing to binding to recipient blood lymphocytes. A significant binding index was taken as greater than 1.0. PBL from diabetic or normal donor BB/W rats were shown to bind significantly to the thyroid gland of 8 out of 19 "potentially diabetic" or diabetic recipient syngeneic rats, but to none of the normal (non diabetic) recipients tested. Sixty percent of "potentially diabetic" BB/W rats and 50% of diabetic rats at 3-4 months of age showed a significant level of binding of donor lymphocytes from syngeneic diabetic or normal animals to their thyroid gland, while, at 5-6 months the proportion of recipient rats giving positive tests was much less (17%). The source of the donor lymphocytes (diabetic, or normal) did not significantly influence the binding. Lymphocyte binding to pancreas was not significantly greater than to control tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of experimental autoimmune thyroiditis in guinea pigs by pretreatment with thyroglobulin-coupled spleen cells

Pretreatment of Strain 2 and Strain 13 guinea pigs with guinea pig thyroglobulin (GPTG) coupled to syngeneic spleen cells (GPTG-SC) suppressed the development of experimental autoimmune thyroiditis (EAT) induced by immunization with GPTG in complete Freund's adjuvant (CFA). Antibody titers to GPTG were only minimally suppressed in GPTG-SC pretreated animals. GPTG-SC also suppressed the sensitization of periotneal exudate T lymphocytes which proliferate in vitro in the presence of GPTG.

The appearance of eosinophils during the development of experimental autoimmune thyroiditis in the guinea pig

A factor chemotactic for eosinophils may be generated by the interaction of a soluble factor derived from antigen-activated lymphocytes with preformed antigen-antibody complexes. The previous demonstration of the in vivo as well as in vitro activity of this factor suggested that it might be of significance in biologic reactions associated with both cell-mediated immunity and antibody formation. Accordingly, guinea pigs were immunized with thyroid extracts in complete Freund's adjuvant to induce thyroiditis, a reaction presumed to be dependent upon cell-mediated immunity for its pathogenesis. Thyroid lesions, consisting of focal lymphoid infiltrates, occurred in the presence or absence of detectable circulating anti-thyroid antibody. With the exception of one guinea pig, eosinophils were found only in the thyroids of those animals with both thyroid lesions and antibody activity. When present, they were located in close proximity to small blood vessels, and in large numbers in the fibrofatty tissue surrounding the gland. Only rarely were they seen in the lymphoid infiltrates themselves. The injection of antibody from these sensitized animals did not lead to the appearance of eosinophils in the thyroids of normal guinea pigs. Thus, in this system, the local accumulation of eosinophils appears to be related to the presence of both lymphocytes in the thyroid and circulating antibody, suggesting that the lymphocyte-derived eosinophil chemotactic factor might be involved in this accumulation.