小鼠自发性自身免疫性甲状腺炎模型的产生
Summary
桥本甲状腺炎的几种动物模型已经建立，NOD小鼠的自发性自身免疫性甲状腺炎也是如此。H-2h4小鼠是一种简单可靠的HT诱导模型。本文介绍了这种方法并评估了病理过程，以便更好地了解SAT小鼠模型。
Abstract
近年来，桥本甲状腺炎（HT）已成为最常见的自身免疫性甲状腺疾病。其特征是淋巴细胞浸润和特异性血清自身抗体的检测。虽然潜在的机制尚不清楚，但桥本甲状腺炎的风险与遗传和环境因素有关。目前，自身免疫性甲状腺炎有几种类型的模型，包括实验性自身免疫性甲状腺炎（EAT）和自发性自身免疫性甲状腺炎（SAT）。
小鼠EAT是HT的常见模型，用脂多糖（LPS）联合甲状腺球蛋白（Tg）或补充完全弗氏佐剂（CFA）进行免疫。EAT小鼠模型在许多类型的小鼠中广泛建立。然而，疾病进展更可能与Tg抗体反应有关，Tg抗体反应在不同的实验中可能有所不同。
SAT也被广泛用于NOD中的HT研究。H-2H4鼠标。点点头。H2h4小鼠是从非肥胖糖尿病（NOD）小鼠与B10杂交中获得的新菌株。A（4R），在有或没有喂食碘的情况下显着诱导HT。在入职过程中，点头。H-2h4小鼠具有高水平的TgAb，伴有甲状腺滤泡组织中的淋巴细胞浸润。然而，对于这种类型的小鼠模型，很少有研究来全面评估诱导碘过程中的病理过程。
本研究建立了用于激素研究的SAT小鼠模型，并评价了长时间碘诱导后的病理变化过程。通过该模型，研究人员可以更好地了解HT的病理发展，并筛选HT的新治疗方法。
Introduction
桥本甲状腺炎（HT），也称为慢性淋巴细胞性甲状腺炎或自身免疫性甲状腺炎，于1912年首次报道1。激素疗法的特征是淋巴细胞浸润和甲状腺滤泡组织损伤。实验室检查主要表现为甲状腺特异性抗体增加，包括抗甲状腺球蛋白抗体（TgAb）和抗甲状腺过氧化物酶抗体（TPOAb）2。激素疗法的发病率在0.4%-1.5%的范围内，占所有甲状腺疾病的20%-25%，近年来该值有所增加3。此外，大量研究报告了激素疗法与甲状腺状癌（PTC）的肿瘤发生和复发有关4，5;潜在的机制仍然存在争议。自身免疫性甲状腺炎也是女性不孕症的重要因素6.因此，激素疗法的发病机制需要明确，为此，稳定简单的动物模型是必不可少的。
为了研究激素疗法的病因，目前的研究采用了两种主要的鼠模型，包括实验性自身免疫性甲状腺炎（EAT）和自发性自身免疫性甲状腺炎（SAT）7，8。用特异性甲状腺抗原（包括粗甲状腺、纯化的甲状腺球蛋白[TG]、甲状腺过氧化物酶[TPO]、重组TPO外域和选定的TPO肽）免疫易感小鼠，以建立EAT鼠模型。此外，佐剂，包括脂多糖（LPS），完全弗氏佐剂（CFA）和其他不寻常的佐剂，在免疫过程中也用于分解免疫耐受9，10，11，12，13，14，15，16，17。
SAT模型是基于NOD研究自身免疫性甲状腺炎自发发展的重要模型。H-2H4小鼠。点点头。H-2h4小鼠是从NOD和B10的杂交中获得的新品系。A（4R）小鼠，然后多次回交至NOD，自身免疫性甲状腺炎易感基因IAk 18，19。点头。H-2h4小鼠不会发展为糖尿病，但自身免疫性甲状腺炎和干燥综合征（SS）的发病率很高19。研究发现，细胞内粘附分子-1（ICAM-1）在NOD的甲状腺组织中高表达。3-4周龄的H-2h4小鼠。而且，随着碘摄入量的增加，甲状腺球蛋白分子的免疫原性增强，进一步上调ICAM-1的表达，ICAM-1在单核细胞浸润过程中起重要作用21。该模型模拟自身免疫过程，同时验证碘剂量与疾病严重程度之间的关系。所建立的方法稳定，成功概率高。SAT模型已应用于诱导自身免疫性甲状腺炎多年，并且仍然是研究自身免疫性甲状腺炎发病机制的有效方法。然而，目前EAT模型的构造方法更加复杂和昂贵;不同的实验室使用不同的免疫方法和注射部位。此外，具有不同遗传背景的小鼠具有不同的诱导率，这需要进一步研究以揭示其有效机制。
然而，SAT模型中甲状腺炎的发展与碘化钠，性别二态性和饲养条件有关。为了揭示SAT模型中自身免疫性甲状腺炎的适当过程，本文描述了不同条件下诱导自身免疫性甲状腺炎的方法。此外，它允许研究自身免疫性甲状腺炎在该疾病不同阶段的发病机制和免疫学进展。
Protocol
Representative Results
Discussion
激素疗法的发生是由于淋巴细胞浸润甲状腺引起的自身免疫系统疾病，进一步损害甲状腺功能，同时产生甲状腺特异性抗体。激素疗法患者的血清TSH、TgAb和TPOAb水平显著升高27。目前，两种主要类型的小鼠模型被广泛用于研究自身免疫性甲状腺炎的病因：EAT和SAT29。EAT小鼠大多使用蛋白质和佐剂进行免疫，以在体内产生异常的免疫环境。这种方法已经使用…
Disclosures
The authors have nothing to disclose.
Acknowledgements
针对人TPO的小鼠单克隆抗体（用作阳性对照）由P. Carayon博士和J. Ruf博士（法国马赛）提供。作者感谢本研究的所有参与者和我们研究团队的成员。这项工作部分得到了中国四川大学华西医院博士后基金（2020HXBH057）和四川省科技支撑计划（项目编号：2021YFS0166）的资助。
Materials
| Butorphanol tartrate | Supelco | L-044 | |
| Dexmedetomidine hydrochloride | Sigma-Aldrich | 145108-58-3 | |
| Enzyme-linked immunosorbent assay (ELISA) well | Sigma-Aldrich | M9410-1CS | |
| Ethanol | macklin | 64-17-5 | |
| Freund’s Adjuvant, Complete | Sigma-Aldrich | F5881 | |
| Freund’s Adjuvant, Incomplete | Sigma-Aldrich | F5506 | |
| Goat anti-Mouse IgG | invitrogen | SA5-10275 | |
| Midazolam solution | Supelco | M-908 | |
| Mouse/rat thyroxine (T4) ELISA | Calbiotech | DKO045 | |
| Paraformaldehyde | macklin | 30525-89-4 | |
| Propidium iodide | Sigma-Aldrich | P4864 | |
| Sodium Iodine | Sigma-Aldrich | 7681-82-5 | |
| Thyroglobulin | Sigma-Aldrich | T1126 | |
| Thyroglobulin ELISA Kit | Thermo Scientific | EHTGX5 | |
| TSH ELISA | Calbiotech | DKO200 | |
| Xylene | macklin | 1330-20-7 |
References
- Ralli, M., et al. Hashimoto’s thyroiditis: An update on pathogenic mechanisms, diagnostic protocols, therapeutic strategies, and potential malignant transformation. Autoimmunity Reviews. 19 (10), 102649 (2020).
- Zhang, Q. Y., et al. Lymphocyte infiltration and thyrocyte destruction are driven by stromal and immune cell components in Hashimoto’s thyroiditis. Nature Communications. 13 (1), 775 (2022).
- Ruggeri, R. M., et al. Autoimmune comorbidities in Hashimoto’s thyroiditis: different patterns of association in adulthood and childhood/adolescence. European Journal of Endocrinology. 176 (2), 133-141 (2017).
- Resende de Paiva, C., Grønhøj, C., Feldt-Rasmussen, U., von Buchwald, C. Association between Hashimoto’s thyroiditis and thyroid cancer in 64,628 patients. Frontiers in Oncology. 7, 53 (2017).
- Ehlers, M., Schott, M. Hashimoto’s thyroiditis and papillary thyroid cancer: are they immunologically linked. Trends in Endocrinology and Metabolism. 25 (12), 656-664 (2014).
- Medenica, S., et al. The role of cell and gene therapies in the treatment of infertility in patients with thyroid autoimmunity. International Journal of Endocrinology. 2022, 4842316 (2022).
- Rose, N. R. The genetics of autoimmune thyroiditis: the first decade. Journal of Autoimmunity. 37 (2), 88-94 (2011).
- Kolypetri, P., King, J., Larijani, M., Carayanniotis, G. Genes and environment as predisposing factors in autoimmunity: acceleration of spontaneous thyroiditis by dietary iodide in NOD.H2(h4) mice. International Reviews of Immunology. 34 (6), 542-556 (2015).
- Terplan, K. L., Witebsky, E., Rose, N. R., Paine, J. R., Egan, R. W. Experimental thyroiditis in rabbits, guinea pigs and dogs, following immunization with thyroid extracts of their own and of heterologous species. The American Journal of Pathology. 36 (2), 213-239 (1960).
- Alexopoulos, H., Dalakas, M. C. The immunobiology of autoimmune encephalitides. Journal of Autoimmunity. 104, 102339 (2019).
- Noviello, C. M., Kreye, J., Teng, J., Prüss, H., Hibbs, R. E. Structural mechanisms of GABA receptor autoimmune encephalitis. Cell. 185 (14), 2469-2477 (2022).
- Pudifin, D. J., Duursma, J., Brain, P. Experimental autoimmune thyroiditis in the vervet monkey. Clinical and Experimental Immunology. 29 (2), 256-260 (1977).
- Esquivel, P. S., Rose, N. R., Kong, Y. C. Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide. The Journal of Experimental Medicine. 145 (5), 1250-1263 (1977).
- Kong, Y. C., et al. HLA-DRB1 polymorphism determines susceptibility to autoimmune thyroiditis in transgenic mice: definitive association with HLA-DRB1*0301 (DR3) gene. The Journal of Experimental Medicine. 184 (3), 1167-1172 (1996).
- Kotani, T., Umeki, K., Hirai, K., Ohtakia, S. Experimental murine thyroiditis induced by porcine thyroid peroxidase and its transfer by the antigen-specific T cell line. Clinical and Experimental Immunology. 80 (1), 11-18 (1990).
- Ng, H. P., Banga, J. P., Kung, A. W. C. Development of a murine model of autoimmune thyroiditis induced with homologous mouse thyroid peroxidase. Endocrinology. 145 (2), 809-816 (2004).
- Ng, H. P., Kung, A. W. C. Induction of autoimmune thyroiditis and hypothyroidism by immunization of immunoactive T cell epitope of thyroid peroxidase. Endocrinology. 147 (6), 3085-3092 (2006).
- Ellis, J. S., Braley-Mullen, H. Mechanisms by which B cells and regulatory T Cells influence development of murine organ-specific autoimmune diseases. Journal of Clinical Medicine. 6 (2), 13 (2017).
- Fang, Y., Yu, S., Braley-Mullen, H. Contrasting roles of IFN-gamma in murine models of autoimmune thyroid diseases. Thyroid. 17 (10), 989-994 (2007).
- Fang, Y., Zhao, L., Yan, F. Chemokines as novel therapeutic targets in autoimmune thyroiditis. Recent Patents on DNA & Gene Sequences. 4 (1), 52-57 (2010).
- Chen, C. R., et al. Antibodies to thyroid peroxidase arise spontaneously with age in NOD.H-2h4 mice and appear after thyroglobulin antibodies. Endocrinology. 151 (9), 4583-4593 (2010).
- Ruf, J., et al. Relationship between immunological structure and biochemical properties of human thyroid peroxidase. Endocrinology. 125 (3), 1211-1218 (1989).
- McLachlan, S. M., Aliesky, H. A., Chen, C. R., Chong, G., Rapoport, B. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a ‘double edged sword’. PLoS One. 7 (9), e43517 (2012).
- McLachlan, S. M., Aliesky, H. A., Chen, C. R., et al. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a ‘double edged sword’[J]. PLoS One. 7 (9), e43517 (2012).
- Hutchings, P. R., et al. Both CD4(+) T cells and CD8(+) T cells are required for iodine accelerated thyroiditis in NOD mice. Cellular Immunology. 192 (2), 113-121 (1999).
- Xue, H., et al. Dynamic changes of CD4+CD25 + regulatory T cells in NOD.H-2h4 mice with iodine-induced autoimmune thyroiditis. Biological Trace Element Research. 143 (1), 292-301 (2011).
- Hou, X., et al. Effect of halofuginone on the pathogenesis of autoimmune thyroid disease in different mice models. Endocrine, Metabolic & Immune Disorders Drug Targets. 17 (2), 141-148 (2017).
- McLachlan, S. M., et al. Dissociation between iodide-induced thyroiditis and antibody-mediated hyperthyroidism in NOD.H-2h4 mice. Endocrinology. 146 (1), 294-300 (2005).
- Danailova, Y., et al. Nutritional management of thyroiditis of hashimoto. International Journal of Molecular Sciences. 23 (9), 5144 (2022).
- Carayanniotis, G. Molecular parameters linking thyroglobulin iodination with autoimmune thyroiditis. Hormones. 10 (1), 27-35 (2011).
- Verginis, P., Li, H. S., Carayanniotis, G. Tolerogenic semimature dendritic cells suppress experimental autoimmune thyroiditis by activation of thyroglobulin-specific CD4+CD25+ T cells. Journal of Immunology. 174 (11), 7433-7439 (2005).
- Flynn, J. C., et al. Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice. Clinical and Experimental Immunology. 137 (3), 503-512 (2004).
- Akeno, N., et al. IFN-α mediates the development of autoimmunity both by direct tissue toxicity and through immune cell recruitment mechanisms. Journal of Immunology. 186 (8), 4693-4706 (2011).
