Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

Summary

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

Abstract

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.

Introduction

Hashimoto's thyroiditis (HT), also known as chronic lymphocytic thyroiditis or autoimmune thyroiditis, was first reported in 1912¹. HT is characterized by lymphocyte infiltration and damage to thyroid follicular tissue. Laboratory tests are mainly manifested as increasing thyroid-specific antibodies, including anti-thyroglobulin antibody (TgAb) and anti-thyroid peroxidase antibody (TPOAb)². The incidence of HT is in the range of 0.4%-1.5%, accounting for 20%-25% of all thyroid diseases, and this value has increased in recent years³. In addition, a large number of studies have reported that HT is associated with the oncogenesis and recurrence of papillary thyroid carcinoma (PTC)^4,5; the potential mechanisms are still controversial. Autoimmune thyroiditis is also an important factor in female infertility⁶. Therefore, the pathogenesis of HT needs to be clear, for which a stable and simple animal model is essential.

To study the etiology of HT, two main kinds of murine models have been employed, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT) in the present studies^7,8. Susceptible mice were immunized with specific thyroid antigens (including the crude thyroid, purified thyroglobulin [TG], thyroid peroxidase [TPO], recombinant TPO ectodomain, and selected TPO peptides) to establish the EAT murine model. In addition, the adjuvants, including lipopolysaccharide (LPS), complete Freund's adjuvant (CFA), and other unusual adjuvants, are also used during the immunization to break down immune tolerance^{9,10,11,12,13,14,15,16,17}.

The SAT model is an important model to study the spontaneous development of autoimmune thyroiditis, which is based on NOD.H-2h4 mice. The NOD.H-2h4 mouse is a new strain obtained from the cross of NOD and B10.A(4R) mice, followed by multiple backcrosses to NOD, with the autoimmune thyroiditis susceptibility gene IAk^18,19. NOD.H-2h4 mice do not develop diabetes, but have a high incidence of autoimmune thyroiditis and Sjogren's syndrome (SS)¹⁹. Studies have found that intracellular adhesion molecule-1 (ICAM-1) is highly expressed in the thyroid tissue of NOD.H-2h4 mice at 3-4 weeks of age. Moreover, with the increase in iodine intake, the immunogenicity of the thyroglobulin molecule is enhanced, which further upregulates the expression of ICAM-1, which plays an important role in the process of monocyte infiltration²¹. This model simulates the autoimmune process while verifying the relationship between iodine dose and disease severity. The established method is stable, with a high probability of success. The SAT model has been applied to induce autoimmune thyroiditis for many years and continues to be an effective method to study the pathogenesis of autoimmune thyroiditis. However, the current construction method of the EAT model is more complicated and expensive; different laboratories use different immunization methods and injection sites. Furthermore, mice with different genetic backgrounds have different rates of induction, which need further study to reveal the potent mechanism.

However, the development of thyroiditis in the SAT model is associated with sodium iodide, sexual dimorphism, and the rearing conditions. To reveal the appropriate procedure of autoimmune thyroiditis in the SAT model, this article described the method of induction of autoimmune thyroiditis in different conditions. In addition, it allows for the study of the pathogenesis and immunological progress of autoimmune thyroiditis in different stages of this disease.

Protocol

The protocol described below was approved by the care and use guidelines established by the Institutional Animal Care and Use Committee of Sichuan University.

1. Preparation

1. House all mice in specific pathogen-free conditions under 12 h light-dark cycles (beginning at 07:00 a.m. and 07:00 p.m., respectively). Maintain the room temperature at 22 °C. Change the bedding materials every week. Provide adequate quantities of standard rodent chow and water.
2. When preparing the stock solution of NaI in water, weigh 2.5 g of the compound and dissolve it in 50 mL of sterile, pure water under vortexing to give a stock solution of 5%. Store this stock solution in a 4 °C freezer, avoiding light, for up to 8 weeks.
3. To prepare a working solution of NaI, draw 2.5 mL of stock solution and dissolve it in 250 mL of regular water as the daily drinking water for NOD.H-2h4 mice, to give a working solution of 0.05%.

2. Induction of thyroiditis

1. SAT model
   1. Prepare the NOD.H-2h4 mice (8 weeks of age) for the study by housing all mice of a single sex (no sexual dimorphism) in barrier cages (five animals per cage) with the feeding conditions described in step 1.1.
   2. To induce autoimmune thyroiditis in NOD.H-2h4 mice, feed all the mice with 0.05% NaI for 8 weeks. Change the NaI water every week and assess the status of the mice on a weekly basis, including appearance, weight, appetite, mental status, and mobility.
2. EAT model
   1. Prepare the BALB/c mice (8 weeks of age) for the study by housing all mice of a single sex (no sexual dimorphism) in barrier cages (five animals per cage) with the feeding conditions described in step 1.1. Feed all mice with 0.05% NaI for 8 weeks.
   2. During the first 2 weeks, subcutaneously inject a mixture of CFA and Tg (200 µL) once a week.
   3. From the third week on, subcutaneously inject a mixture of IFA and Tg (200 µL) once a week for 3 weeks.

3. Measurements

1. Preparation of peripheral blood samples
   1. After the induction, anesthetize the mice with a volume of 0.01 mL/g anesthetic by intraperitoneal injection. Prepare the anesthetic by mixing midazolam (40 µg/100 µL for sedation), medetomidine (7.5 µg/100 µL for sedation), and butorphanol tartrate (50 µg/100 µL for analgesia) in phosphate-buffered saline (PBS).
      NOTE: The specific concentrations of each component in the anesthesia mixture are: midazolam 13.33µg/100µL, medetomidine 2.5µg/100µL, and butorphanol 16.7µg/100µL. For specific dosages used in mice, the doses are: midazolam 4µg/g, medetomidine 0.75µg/g, and butorphanol 1.67µg/g. Anesthesia depth was confirmed when the mouse's limb muscles relaxed, the whiskers had no touch response, and there was loss of  pedal reflex.
   2. After the mice are anesthetized, prepare the peripheral blood samples, by fixing the mouse with one hand and pressing the eye skin to protrude the eyeball. Then, insert the capillary tube into the inner corner of the eye and penetrate at a 30-45 degree angle to the plane of the nostril. Apply pressure while gently rotating the capillary tube. Blood will flow into the tube via capillary action.
   3. Put the blood into a 4 °C refrigerator for 2 h and then centrifuge at 1,000 × g for 10 min at 4 °C to get the sample. For further analysis, store the rest of the sample at -80 °C.
2. Preparation of thyroid tissue samples
   1. Humanely euthanize the animal according to the institutional policies. Then, dissect the chest wall to expose the heart, cut open the right atrium, and infuse saline into the left ventricle by an intravenous infusion needle attached to a 20 mL syringe until the tissue turns white.
   2. Fix the mouse on the dissection table with pins. Sterilize the neck with 75% ethanol. Cut the skin of the mouse along the median line of the neck from the top of the sternum to the lower jaw with tissue scissors to completely expose the neck tissue.
   3. Observe the pair of pink glands, the submandibular glands, below which is the anterior trachea muscle. Separate the glands and muscle with ophthalmic scissors and forceps to expose the trachea and thyroid cartilage.
   4. Separate the tissue under the cartilage, using curved forceps to pick up the cartilage and trachea together. Cut the distal and proximal ends of the cartilage and trachea, respectively, and remove the thyroid gland together with the trachea.
   5. Immerse the glands in 4% paraformaldehyde for 12-24 h. Then, transfer the tissue to 70% ethanol.
   6. Place the individual lobes of thyroid biopsy material in the processing cassettes and dehydrate through the following serial alcohol gradient: 70%, 80%, 90%, 95%, 100%, 100%, ethanol for 40 min each; 1/2 xylene + 1/2 absolute ethanol for 2 h; 100% xylene for 1.5 h; 100% xylene for 1.5 h; 1/2 xylene + 1/2 paraffin for 2 h; oven at 40 ° C for 40 min; paraffin I for 30 min; and paraffin II for 30 min. Embed them in paraffin wax blocks.
   7. Before the staining, dewax 5 µm thick thyroid tissue sections in xylene (as follows) and rehydrate through the following decreasing concentrations of ethanol: xylene I for 10 min; xylene II for 10 min; xylene III for 10 min; absolute ethanol I for 5 min; absolute ethanol II for 5 min; 90% alcohol for 5 min; 80% alcohol for 5 min; 70% alcohol for 5 min; and 50% alcohol for 5 min.
   8. Stain the tissues with hematoxylin and eosin (H&E).Stain with hematoxylin for 15 min, rinse with tap water for 15 min, add 1% hydrochloric acid ethanol for 10 s, rinse with running water, and then with 50%, 70%, and 80% ethanol, each for 3 min. Perform 0.5% eosin ethanol staining for 2 min and rinse with running water. Place the paraffin sections successively in 75% ethanol for 2 min, 85% ethanol for 2 min, absolute ethanol for 5 min, and xylene for 5 min. Fix the slices with neutral gum and glass slides.
   9. To evaluate the extent of lymphocytic thyroiditis, score the thyroid sections as follows: 0: little or no lymphocyte infiltration in the thyroid tissue; 1+: more than 1/8 of the gland is infiltrated in one or several foci; 2+: no more than 1/4 of the gland is infiltrated with lymphocytes; 3+: 1/4-1/2 of the gland is infiltrated with lymphocytes; 4+: more than 1/2 of the gland is destroyed.
      NOTE: It is recommended to remove the thyroid cartilage to keep the whole structure of the mouse thyroid, which is too small to remove from the trachea.
3. Measurement of TPOAb
   NOTE: Chinese hamster ovary (CHO) cells stably expressing mouse TPO were established in our lab. Mouse TPO cDNA was excised by XbaI and NheI. Then, the cDNA was transferred to pcDNA5/FRT. After the plasmid was constructed successfully, it was transfected into the CHO cells and selected with hygromycin B (100 g/mL).
   1. After the construction of mTPO-CHO cells, dilute the mouse sera from step 3.1.3 (1:50) and incubate with the mTPO-CHO cells for 2 h. After incubation, exclude cell staining with propidium iodide (1 g/mL) and analyze the sample by flow cytometry using fluorescein isothiocyanate-conjugated, affinity-purified goat anti-mouse IgG. Screen surviving single-cell populations by FSC-A and SSC-A channels and select FITC- and PI-tagged cells from the single-cell populations²¹.
   2. Include the serum of unimmunized BALB/c or C57BL/6 mice bound to IgG as the negative control. Include mouse monoclonal antibodies to human TPO²² as positive controls that recognize mouse TPO²³. Express TPO binding data as geometric means.
4. Measurement of TgAb
   NOTE: Use an ELISA kit to detect thyroglobulin by following the manufacturer's instructions.
   1. Dilute the standard samples in the microcentrifuge tube according to the instructions. Take the kit out from the refrigerator and keep it at room temperature for 30 min.
   2. Set the standard wells, blank wells, and testing wells. Blank wells only get buffer, while standard wells get standard solutions. Add 10 µL of samples into the testing wells. When adding the samples, try not to touch the walls of the wells, and shake the plate gently to move the samples to the bottom of the wells.
   3. Include serum from BALB/c mice immunized with Tg, CFA, and IFA²⁴ as the positive control and serum from 8-week-old NOD.H-2h4 mice on regular water as the negative control.
   4. Incubate the samples in a 37 °C water bath for 30 min. Dilute the washing buffer with distilled water at a ratio of 1:30. Then, remove the sealing film, shake off the liquid inside the enzyme plate and pat dry on absorbent paper, and add 350 µL of washing buffer for 30 s. Repeat this procedure five times and then pat the wells dry.
   5. Add 50 µL of enzyme labeling reagent to each well, except for the blank wells, and incubate in a 37 ° water bath for 30 min. Remove the sealing film, shake off the liquid inside the enzyme plate, and pat dry on absorbent paper.
   6. Add 50 µL of developer A to each well followed by 50 µL of developer B and gently shake the wells to mix for 5 min. Add 50 µL of the termination solution in each well for 15 min to stop the reaction. Measure the absorbance (OD) of each well at a wavelength of 450 nm using a microplate reader. Present the TgAb data as the optical density (OD) at 490 nm.
5. Measurement of T4 and thyroid stimulating hormone (TSH)
   NOTE: Measure T4 and TSH levels (in 10 µL aliquots) by ELISA by following the manufacturer's instructions.
   1. Dilute the enzyme conjugate to 1:11 with the assay diluent; dilute the samples to be measured (1:100) using 0.01 M PBS.
   2. Add 10 µL of the standard and the sample to the corresponding wells, add 100 µL of enzyme conjugate to each well, and incubate at room temperature for 1 h.
   3. Discard the liquid in the wells and clean it with washing buffer three times.
   4. Add 100 µL of 3,3',5,5'-tetramethyl benzidine (TMB) and incubate at room temperature for 15 min.
   5. Add 50 µL of stop solution and mix gently for 15-20 s.
   6. Determine the absorbance (OD) using a microplate reader at a wavelength of 450 nm and calculate the sample concentration.
   7. Use statistical software for performing t-test or rank sum test and plot the results.

Results

The histological changes were strikingly different in female and males, the duration of iodine intake, and the solution of NaI. As shown in Figure 1, ~10% of NOD.H-2h4 mice developed SAT even without iodine induction at the age of 24 weeks, and all the mice eventually developed thyroiditis. When given regular water, there was no significant difference in the histological changes between males and females. The addition of NaI to the drinking water accelerated the development of thyroiditis. I...

Discussion

HT occurs due to an autoimmune system disorder caused by lymphocytes infiltrating the thyroid gland, further impairing thyroid function, while producing thyroid-specific antibodies. Serum TSH, TgAb, and TPOAb levels in HT patients are significantly elevated²⁷. At present, two main kinds of murine models are widely used to study the etiology of autoimmune thyroiditis: EAT and SAT²⁹. EAT mice are mostly immunized using proteins and adjuvants to create an abnormal immune envir...

Materials

Name	Company	Catalog Number	Comments
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