Experimental Autoimmune Thyroiditis in the Mouse

Yi‐chi M. Kong1

1 Wayne State University School of Medicine, Detroit, Michigan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.7
DOI:  10.1002/0471142735.im1507s78
Online Posting Date:  August, 2007
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Abstract

Experimental autoimmune thyroiditis (EAT) in mice is an excellent model for Hashimoto's thyroiditis (HT). It is induced with thyroglobulin (Tg), a known thyroid autoantigen that is common to both mouse and human and for which several conserved, thyroiditogenic epitopes have been identified. This unit describes induction and evaluation of EAT using thyroid histology and in vitro proliferative response assays. An ELISA is presented to detect the level of antibody to mouse thyroglobulin (MTg). To induce EAT, either bacterial lipopolysaccharide (LPS) or supplemented complete Freund's adjuvant (CFA) can be used as adjuvant. A support protocol for preparing MTg is included. The T cell proliferation assay can be used to examine the antigenicity of synthetic peptides derived from MTg or heterologous Tg. EAT can be adoptively transferred utilizing cells that have been expanded in vitro, as described. A protocol is provided for inducing tolerance using deaggregated MTg; induction of tolerance requires larger amounts of MTg but efficiently suppresses EAT development. Also included is a protocol to demonstrate the role of regulatory T cells in mediating tolerance. A protocol to delineate HLA association with HT is illustrated using HLA class II transgenic mice. Curr. Protoc. Immunol. 78:15.7.1‐15.7.21. © 2007 by John Wiley & Sons, Inc.

Keywords: autoimmune thyroiditis; experimental autoimmune thyroiditis; EAT; HLA class II transgenes; HLA‐DR3 association; regulatory T cells in EAT

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction and Evaluation of Experimental Autoimmune Thyroiditis
  • Alternate Protocol 1: Induction of Experimental Autoimmune Thyroiditis Using CFA as Adjuvant
  • Support Protocol 1: Preparation of Mouse Thyroglobulin
  • Support Protocol 2: Measurement of T Cell Proliferative Response to Mouse Thyroglobulin
  • Basic Protocol 2: Adoptive Transfer of Experimental Autoimmune Thyroiditis
  • Basic Protocol 3: Induction of Tolerance to Experimental Autoimmune Thyroiditis
  • Basic Protocol 4: Demonstration of Regulatory T Cell Control of Tolerance to Experimental Autoimmune Thyroiditis
  • Basic Protocol 5: Determination of HLA Association with Autoimmune Thyroiditis in Class II Transgenic Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction and Evaluation of Experimental Autoimmune Thyroiditis

  Materials
  • Specific pathogen–free, susceptible female mice (see Table 15.7.1), 8 to 12 weeks old
  • Acidified, chlorinated water (see recipe)
  • 400 µg/ml mouse thyroglobulin (MTg) in nonpyrogenic saline (see protocol 3)
  • 200 µg/ml trichloroacetic acid (TCA)–precipitated Salmonella enteritidis lipopolysaccharide (LPS) in saline (Difco or Sigma)
  • Formalin fixative: 10% (v/v) formalin in 0.15 M sodium acetate
  • 1‐ml syringes with 26‐G needles
  • Histology sample cassettes
  • Iris scissors
  • Small forceps
  • Additional reagents and equipment for collecting serum (unit 1.7), injection of mice (unit 1.6), euthanizing animals (unit 1.8), staining tissue sections (unit 21.4), performing T cell proliferative assay (see protocol 4), and performing indirect ELISA (unit 2.1)
    Table 5.7.1   MaterialsCommonly Used Mouse Strains for EAT

    Susceptible Resistant
    Conventional strains
    H2k CBA/J a H2b C57BL/10 (B10)
    C57BR C57BL/6 (B6)
    B10.K, B10.BR H2d BALB/c
    H2s SJL B10.D2
    B10.S H2f B10.M
    H2q B10.Q b
    HLA class II transgenic strains c
    DRB1*0301 (DR3) a DRB1*1502 (DR2)
    DRB1*0401 (DR4)
    DQA1*0301/DQB1*0302 (DQ8) d

     aMost commonly used strain.
     bIntermediate in susceptibility.
     cGenerated on Ab0, AE0, or Ab0/NOD background.
     dWeakly susceptible to EAT induction with human thyroglobulin.

Alternate Protocol 1: Induction of Experimental Autoimmune Thyroiditis Using CFA as Adjuvant

  • 800 µg/ml mouse thyroglobulin (MTg) in PBS, pH 7.2, or nonpyrogenic saline (see protocol 3)
  • Supplemented complete Freund's adjuvant (CFA; see recipe)
  • Two 5‐ or 10‐ml Luer‐Lok glass syringes
  • 3‐way disposable stopcock
  • Small beaker with water
  • 1‐ml Luer‐Lok glass syringe and 25‐G needles
  • Additional reagents and equipment for evaluating the response to antigen (see protocol 1, steps 4 to 11)
NOTE: As with all animal protocols, the institutional animal care and use committee (IACUC) must preapprove this protocol. The IACUC may object to administering two injections with CFA; however, administering two priming doses 7 days apart produces no ill effects in mice and is essential to induction of autoimmune disease with a self antigen such as MTg.

Support Protocol 1: Preparation of Mouse Thyroglobulin

  Materials
  • 300 to 500 mice, 2 to 8 months old
  • Sephadex G‐200
  • PBS, pH 7.2 ( appendix 2A), 4°C
  • 5‐ml plastic tubes
  • 2.5 × 90–cm column, fraction collector, 500‐ml buffer reservoir and glass collection tubes, 4°C
  • Small straight forceps
  • No. 5, 90°‐angle curved forceps (Roboz Surgical Instruments)
  • 35‐mm plastic petri dishes
  • 15‐ml glass tissue homogenizer, 4°C
  • Ultracentrifuge with SW50.1 rotor and 5‐ml ultracentrifuge tubes
  • Additional reagents and equipment for column chromatography ( appendix 3I)

Support Protocol 2: Measurement of T Cell Proliferative Response to Mouse Thyroglobulin

  Materials
  • MTg‐immunized (see protocol 1 or protocol 2) and control mice
  • Complete RPMI‐1% NMS (see recipe), freshly prepared
  • 400 µg/ml MTg (see protocol 3) in RPMI, filter sterilized
  • Accessory cells for lymph node cell culture: spleen cells isolated from normal mice and irradiated with 2000 rads
  • 96‐well microtiter plate
  • Additional reagents and equipment for preparing single‐cell suspensions of spleen or lymph node cells (unit 3.1), counting viable cells by trypan blue dye exclusion ( appendix 3B), and labeling and harvesting cells ( appendix 3D)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Adoptive Transfer of Experimental Autoimmune Thyroiditis

  Materials
  • Donors: mice immunized with MTg and LPS (see protocol 1), 14 to 28 days after immunization (for spleen cells), or mice immunized with MTg emulsified in CFA (see protocol 2), 12 to 21 days after immunization (for lymph node cells)
  • Complete RPMI‐1% NMS (see recipe)
  • 400 µg/ml MTg (see protocol 3) in RPMI, filter‐sterilized
  • Accessory cells for lymph node cell cultures: spleen cells from unimmunized mice, irradiated with 2000 rad
  • Recipients: normal female syngeneic mice
  • 75‐cm2 tissue culture flasks
  • Beckman TJ‐6 refrigerated centrifuge and TH‐4 rotor (or equivalent)
  • 1‐ml syringe with 26‐G needle
  • Additional reagents and equipment for preparing single‐cell suspensions of spleen and lymph node cells (unit 3.1), counting viable cells by trypan blue dye exclusion ( appendix 3B), injection of mice (unit 1.6) and histologic evaluation of thyroids (see protocol 1, steps 5 to 9)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Induction of Tolerance to Experimental Autoimmune Thyroiditis

  Materials
  • Female mice, 6 to 7 weeks old
  • 1.5 to 2 mg/ml MTg in PBS (see protocol 3)
  • PBS, pH 7.2 ( appendix 2A) or nonpyrogenic saline (McGaw)
  • 0.8‐ml ultracentrifuge tubes (5 × 41–mm) and adaptor for 5‐ml rotor slot
  • Ultracentrifuge with SW 50.1 rotor
  • Small forceps
  • 1‐ml syringe with 26‐G needle
  • Additional reagents and equipment for inducing EAT (see protocol 1, steps 1 to 3)

Basic Protocol 4: Demonstration of Regulatory T Cell Control of Tolerance to Experimental Autoimmune Thyroiditis

  Materials
  • Female susceptible mice, 6‐ to 7‐weeks old
  • 1.5 to 2 mg/ml MTg in PBS (see protocol 3)
  • CD25 MAb: rat IgG1 λ, clone PC61 hybridoma (ATCC #TIB‐222), purified from ascites fluid or hollow fiber reactor cell culture system (unit 2.6) and diluted in nonpyrogenic saline for injection
  • Control rat IgG
  • Fluorescently conjugated CD4, CD8, and CD25 MAbs for analysis by flow cytometry to verify depletion of CD4+CD25+ T cells (also see Chapter 5)
  • Additional reagents and equipment for induction of tolerance ( protocol 6, steps 1 to 6), induction of EAT ( protocol 6, step 7), evaluation of thyroids for EAT ( protocol 6, step 8), intravenous injection into the tail vein of mice (unit 1.6), obtaining peripheral blood leukocytes from mice (unit 3.1), flow cytometry (Chapter 5), and euthanasia of mice (unit 1.8)

Basic Protocol 5: Determination of HLA Association with Autoimmune Thyroiditis in Class II Transgenic Mice

  Materials
  • DR3, DR2, DQ8, DR3/DQ8 transgenic Ab0 or AE0 mice, either sex, 2 to 4 months old
  • Fluorescently conjugated MAbs (also see Chapter 5): DRB1‐specific (clone L227, ATCC #HB‐96) and DQ‐specific (clone IVD12, ATCC #HB‐144)
  • 1.5 to 2 mg/ml MTg (see protocol 3) or HTg (use 5 to 10 g normal thyroid from necropsy, prepared as in protocol 3)
  • Additional reagents and equipment for obtaining peripheral blood leukocytes from mice (unit 3.1), flow cytometry (Chapter 5), intravenous tail vein injection of mice (unit 1.6), and induction and evaluation of EAT ( protocol 1)
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Figures

Videos

Literature Cited

   Beisel, K.W., David, C.S., Giraldo, A.A., Kong, Y.M., and Rose, N.R. 1982a. Regulation of experimental autoimmune thyroiditis: Mapping of susceptibility to the I‐A subregion of the mouse H‐2. Immunogenetics 15:427‐430.
   Beisel, K.W., Kong, Y.M., Babu, K.S.J., David, C.S., and Rose, N.R. 1982b. Regulation of experimental autoimmune thyroiditis: Influence of non‐H‐2 genes. J. Immunogenet. 9:257‐265.
   Caturegli, P., Vidalain, P.O., Vali, M., Aguilera‐Galaviz, L.A., and Rose, N.R. 1997. Cloning and characterization of murine thyroglobulin cDNA. Clin. Immunol. Immunopathol. 85:221‐226.
   ElRehewy, M., Kong, Y.M., Giraldo, A.A., and Rose, N.R. 1981. Syngeneic thyroglobulin is immunogenic in good responder mice. Eur. J. Immunol. 11:146‐151.
   Esquivel, P.S., Rose, N.R., and Kong, Y.M. 1977. Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide. J. Exp. Med. 145:1250‐1263.
   Flynn, J.C., McCormick, D.J., Brusic, V., Wan, Q., Panos, J.C., Giraldo, A.A., David, C.S., and Kong, Y.M. 2004. Pathogenic human thyroglobulin peptides in HLA‐DR3 transgenic mouse model of autoimmune thyroiditis. Cell. Immunol. 229:79‐85.
   Flynn, J.C., Meroueh, C., Snower, D.P., David, C.S., and Kong, Y.M. 2007. Depletion of CD4+CD25+ regulatory T cells exacerbates sodium iodide–induced experimental autoimmune thyroiditis in human leucocyte antigen DR3 (DRB1*0301) transgenic class II‐knock‐out non‐obese diabetic mice. Clin. Exp. Immunol. 147:547‐554.
   Kong, Y.M. 1998. Animal models of autoimmune thyroiditis: Recent advances. In Endocrine Autoimmunity and Associated Conditions (A.P. Weetman, ed.) pp. 1‐23. Kluwer Academic Publishers BV, Dordrecht, The Netherlands.
   Kong, Y.M., David, C.S., Giraldo, A.A., ElRehewy, M., and Rose, N.R. 1979. Regulation of autoimmune response to mouse thyroglobulin: Influence of H‐2D‐end genes. J. Immunol. 123:15‐18.
   Kong, Y.M., Okayasu, I., Giraldo, A.A., Beisel, K.W., Sundick, R.S., Rose, N.R., David, C.S., Audibert, F., and Chedid, L. 1982. Tolerance to thyroglobulin by activating suppressor mechanisms. Ann. N. Y. Acad. Sci. 392:191‐209.
   Kong, Y.M., Giraldo, A.A., Waldmann, H., Cobbold, S.P., and Fuller, B.E. 1989. Resistance to experimental autoimmune thyroiditis: L3T4+ cells as mediators of both thyroglobulin‐activated and TSH‐induced suppression. Clin. Immunol. Immunopathol. 51:38‐54.
   Kong, Y.M., McCormick, D.J., Wan, Q., Motte, R.W., Fuller, B.E., Giraldo, A.A., and David, C.S. 1995. Primary hormonogenic sites as conserved autoepitopes on thyroglobulin in murine autoimmune thyroiditis: Secondary role of iodination. J. Immunol. 155:5847‐5854.
   Kong, Y.M., Lomo, L.C., Motte, R.W., Giraldo, A.A., Baisch, J., Strauss, G., Hammerling, G.J., and David, C.S. 1996. HLA‐DRB1 polymorphism determines susceptibility to autoimmune thyroiditis in transgenic mice: Definitive association with HLA‐DRB1*0301 (DR3) gene. J. Exp. Med. 184:1167‐1172.
   Kong, Y.M., Flynn, J.C., Wan, Q., and David, C.S. 2003. HLA and H2 class II transgenic mouse models to study susceptibility and protection in autoimmune thyroid disease. Autoimmunity 36:397‐404.
   Lewis, M., Giraldo, A.A., and Kong, Y.M. 1987. Resistance to experimental autoimmune thyroiditis induced by physiologic manipulation of thyroglobulin level. Clin. Immunol. Immunopathol. 45:92‐104.
   Lewis, M., Fuller, B.E., Giraldo, A.A., and Kong, Y.M. 1992. Resistance to experimental autoimmune thyroiditis is correlated with the duration of raised thyroglobulin levels. Clin. Immunol. Immunopathol. 64:197‐204.
   Li, H.S., Jiang, H.Y., and Carayanniotis, G. 2007. Modifying effects of iodine on the immunogenicity of thyroglobulin peptides. J. Autoimmun. 28:171‐176.
   Morris, G.P. and Kong, Y.M. 2006. Tolerance to autoimmune thyroiditis: CD4+CD25+ regulatory T cells influence susceptibility but do not supersede MHC class II restriction. Front. Biosci. 11:1234‐1243.
   Morris, G.P., Chen, L., and Kong, Y.M. 2003. CD137 signaling interferes with activation and function of CD4+CD25+ regulatory T cells in induced tolerance to experimental autoimmune thyroiditis. Cell. Immunol. 226:20‐29.
   Morris, G.P., Yan, Y., David, C.S., and Kong, Y.M. 2005. H2A‐ and H2E‐derived CD4+CD25+ regulatory T cells: A potential role in reciprocal inhibition by class II genes in autoimmune thyroiditis. J. Immunol. 174:3111‐3116.
   Nabozny, G.H., Simon, L.L., and Kong, Y.M. 1990. Suppression in experimental autoimmune thyroiditis: The role of unique and shared determinants on mouse thyroglobulin in self‐tolerance. Cell. Immunol. 131:140‐149.
   Okayasu, I., Kong, Y.M., David, C.S., and Rose, N.R. 1981. In vitro T‐lymphocyte proliferative response to mouse thyroglobulin in experimental autoimmune thyroiditis. Cell. Immunol. 61:32‐39.
   Rose, N.R., Twarog, F.J., and Crowle, A.J. 1971. Murine thyroiditis: Importance of adjuvant and mouse strain for the induction of thyroid lesions. J. Immunol. 106:698‐704.
   Simon, L.L., Krco, C.J., David, C.S., and Kong, Y.M. 1985. Characterization of the in vitro murine T‐cell proliferative responses to murine and human thyroglobulins in thyroiditis‐susceptible and ‐resistant mice. Cell. Immunol. 94:243‐253.
   Simon, L.L., Justen, J.M., Giraldo, A.A., Krco, C.J., and Kong, Y.M. 1986. Activation of cytotoxic T cells and effector cells in experimental autoimmune thyroiditis by shared determinants of mouse and human thyroglobulins. Clin. Immunol. Immunopathol. 39:345‐356.
   Vladutiu, A.O. and Rose, N.R. 1971. Autoimmune murine thyroiditis: Relation to histocompatibility (H‐2) type. Science 174:1137‐1139.
   Wan, Q., Shah, R., McCormick, D.J., Lomo, L.C., Giraldo, A.A., David, C.S., and Kong, Y.M. 1999. H2‐E transgenic class II‐negative mice can distinguish self from nonself in susceptibility to heterologous thyroglobulins in autoimmune thyroiditis. Immunogenetics 50:22‐30.
   Wei, W.‐Z., Jacob, J.B., Zielinski, J.F., Flynn, J.C., Shim, K.D., Alsharabi, G., Giraldo, A.A., and Kong, Y.M. 2005. Concurrent induction of antitumor immunity and autoimmune thyroiditis in CD4+CD25+ regulatory T cell‐depleted mice. Cancer Res. 65:8471‐8478.
Key References
   Kong, 1998. See above.
  An overview of murine EAT as a model to study human autoimmune disease.
   Kong et al., 1996. See above.
  First HLA class II transgenic study in EAT, showing use of proper control groups.
   Kong et al., 2003. See above.
  A review of susceptibility and protection in EAT using HLA and H2 transgenic models.
   Morris and Kong, 2006. See above.
  Outline the history of characterization of regulatory T cells in EAT as immunologic techniques advanced.
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