Contrasting Roles of IFN-c in Murine Modelsof Autoimmune Thyroid Diseases

Yujiang Fang,1,* Shiguang Yu,1,* and Helen Braley-Mullen1–3

Interferon-gamma (IFN-g), a prototypic proinflammatory cytokine produced by several different cell types, in-cluding the Th1 subset of CD4+ T cells, plays an important role in inflammation and autoimmune diseases. Thisreview focuses on the varied and often contrasting roles of IFN-g in three murine models of autoimmune thyroiddisease, experimentally induced autoimmune thyroiditis, the model of iodine-induced spontaneous autoimmunethyroiditis in NOD.H-2h4mice and several different murine models of Graves’ disease.

Introduction

Interferon-gamma (IFN-g) is a multifunctional cyto-kine that plays an important role in many autoimmune

diseases, including thyroiditis. IFN-g is the prototypic Th1cytokine produced by CD4þ Th1 cells, and it is also producedby CD8þ T cells and natural killer (NK) cells (1). IFN-g in-duces MHC class I and II on antigen presenting cells (APCs)and other cells, upregulates adhesion molecules as well ascertain chemokines and chemokine receptors to recruit Tcells to sites of inflammation, activates macrophages, andpromotes IgG2A antibody production (1). Neutralization orgenetic ablation of IFN-g or the IFN-g receptor (IFN-gR) canhave both positive and negative effects on many autoim-mune diseases, including autoimmune thyroid diseases. Inthis brief review, we discuss the various and often con-trasting effects of neutralization, transgenic overexpression,or genetic ablation of IFN-g, IFN-gR, and interleukin (IL)-12 in mouse models of autoimmune thyroid diseases.

Autoimmune thyroid diseases encompass several condi-tions that have in common cellular and humoral immuneresponses targeted to the thyroid gland. The most commonautoimmune thyroid diseases in humans are Graves’ disease(GD) and Hashimoto’s thyroiditis (2). While reviewing therole of IFN-g in autoimmune thyroid diseases, the primaryfocus will be on the models of experimental autoimmunethyroiditis (EAT) in mice and the iodine-induced model ofspontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4mice. Although both animal models are often consideredto represent animal models of Hashimoto’s thyroiditis, theeffects of IFN-g ablation or neutralization on EAT and SATare very different, suggesting basic differences in underlyingmechanisms in these two animal models.

Experimental Autoimmune Thyroiditis

EAT is an organ-specific autoimmune disease inducible ingenetically susceptible strains of mice by injection of mousethyroglobulin (MTg) and adjuvant (active immunization) (2–4), or by transfer of MTg-primed donor spleen cells activatedwith MTg in vitro (adoptive transfer model) (5,6). Thyroidlesions in both models of EAT are characterized by infiltra-tion of the thyroid by mononuclear cells, including CD4þ

and CD8þ T cells, plasma cells, and macrophages (2–6). In-flammation tends to be long lasting (chronic), most miceproduce anti-Tg antibodies, and they typically have normalserum thyroxine (T4) levels throughout the course of disease(3,5). CD4þ T cells are the primary effector cells for both theactive immunization and adoptive transfer EAT models (7,8).CD8þ T cells reportedly have effector function in some activeimmunization models of EAT (3,9) but not in the adoptivetransfer model studied in our laboratory (7,10).

Granulomatous (G)-EAT

A severe and histologically distinct granulomatous formof EAT (G-EAT) studied extensively in our laboratory is in-duced when cells from MTg-sensitized donors are activatedin vitro with MTg and IL-12 or with MTg and anti-IL-2Rmonoclonal antibody (mAb) (7,11,12). Thyroid lesions inG-EAT, like those in EAT, are characterized by infiltration ofthe thyroid by CD4þ and CD8þ T cells, plasma cells, andmacrophages. In the adoptive transfer model studied in ourlaboratory, inflammation in G-EAT is more severe than inEAT, and there are thyroid epithelial cell (TEC) proliferation,large numbers of histocytes, multinucleated giant cells, fibro-sis, and variable numbers of neutrophils, in addition tomono-nuclear infiltration (7,11,12). CD4þ T cells are the primary

Departments of 1Internal Medicine and 2Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri.3Department of Veterans Affairs, Harry S. Truman VA Medical Center, Columbia, Missouri.*Yujiang Fang and Shiguang Yu contributed equally to this review.

THYROIDVolume 17, Number 10, 2007ª Mary Ann Liebert, Inc.DOI: 10.1089=thy.2007.0261

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effector cells for G-EAT (7,12). CD8þ T cells are not requiredfor effector function in G-EAT, but function primarily topromote resolution of thyroid lesions (7,10). Thyroid in-flammation reaches maximal severity 19–21 days after celltransfer. Inflammation then spontaneously resolves if CD8þ

T cells are present, or when essentially all thyroid follicles aredestroyed, there is ongoing inflammation and developmentof fibrosis. Mice with these very severe lesions and fibrosisusually have low serum T4 (7,12–14). While reviewing therole of IFN-g in experimentally induced models of thyroiditisin mice, EAT and G-EAT will be considered together, andareas where there may be differences and=or where infor-mation is known for only one model will be mentioned.

Role of IFN-c in EAT and G-EAT

As an important Th1 cytokine (15), IFN-g plays an importantrole in immune and inflammatory responses, and it has beenshown to both promote and suppress development of EAT inmice. In some early studies, injection of IFN-g into thyroids ofCBA=J mice promoted development of EAT and productionof anti-MTg autoantibodies, and development of EAT wasinhibited by anti-IFN-g mAb (16–19). These results all suggestthat IFN-g plays an important role in thyroid destruction inEAT. However, this idea is challenged by other results show-ing that systemic administration of IFN-g suppresses EAT (20),and mice unable to respond to IFN-g (IFN-gR–=– mice) (21) orproduce IFN-g (IFN-g–=– mice) [(22) and our unpublished re-sults] are susceptible to EAT. In addition, interferon regulatoryfactor-1 (IRF-1) is an important regulator of IFN-g-mediatedimmune reactions, and IRF-1–=– NOD mice develop EAT andanti-MTg antibodies comparable to IRF-1þ=þ and IRF-1þ=–

mice (23). In the adoptive transfer model of G-EAT, neutrali-zation of IFN-g during in vitro activation of effector cells withMTg (no exogenous cytokines) promotes development ofmoresevere EAT with granulomatous histology (24), and spleno-cytes from IFN-g–=– mice activated with MTg and IL-12 trans-fer severe G-EAT to IFN-g–=– recipients (22).

The fact that IFN-gR–=–, IFN-g–=–, and IRF-1–=– mice candevelop EAT and that IFN-g–=– mice can develop severeG-EAT clearly indicates that IFN-g is not absolutely requiredfor induction of EAT. However, IFN-gR–=– mice have a milderdisease phenotype and thyroid lesions that resolve earlierthan in wild-type (WT) controls (21). In our studies, G-EATdiffers histologically in WT and IFN-g–=– mice (22), and in theabsence of IFN-g, there is earlier resolution of G-EAT and lessfibrosis (13,14). Specifically, thyroids of IFN-g–=– recipients ofIFN-g–=– effector cells have many eosinophils infiltrating thethyroids, they develop minimal fibrosis, and thyroid lesionsresolve considerably earlier than in WT mice (13,22). In con-trast, thyroids ofWT recipients ofWT effector cells havemanyneutrophils and almost no eosinophils, they develop exten-sive fibrosis, and inflammation is prolonged (13,22,25).Compared with WT mice, thyroids of IFN-g–=– recipients ofIFN-g–=– effector cells with severe G-EAT have lower mRNAexpression of Th1 cytokines and proinflammatory media-tors such as tumor necrosis factor-� (TNF-�) and induciblenitric oxide synthetase (iNOS), and higher expression of mostTh2 cytokines, particularly IL-10 (13,22). In WT mice, ex-pression of IFN-g and of other proinflammatory cytokinessuch as TNF-� and IL-17 is generally higher in thyroids withdelayed resolution and fibrosis, while Th2 cytokines such as

IL-10, IL-4, and IL-13 are generally higher in thyroids withearly resolution and less fibrosis (13,25). IFN-g can inducenecrosis or cooperate in the presence of TNF-� to inducetissue damage by apoptosis of epithelial cells (26). Increasedapoptosis of TEC and necrosis are evident in thyroids thatexpress high amounts of IFN-g and where inflammationprogresses to fibrosis (13,14), and IFN-g and other proin-flammatory cytokines can promote TEC apoptosis and moredestructive thyroiditis in mice (27,28). IFN-g also modulateschemokine production, including IFN-g-inducible protein 10(IP-10)=CXC chemokine ligand 10 (CXCL10), monokine in-duced by IFN-g (Mig)=CXCL9, and IFN-g-inducible T cell-� chemoattractant=CXCL11. These and other chemokines areimportant in promoting migration of lymphocytes to sites ofinflammation in thyroiditis and other autoimmune diseases(29–36), indicating that IFN-g plays an important role incontrolling leukocyte migration. Thus, IFN-g plays a criticalrole in regulating the inflammatory response in G-EAT,promoting chronic inflammation and fibrosis, and inhibitingresolution of thyroid lesions. The profibrotic function of IFN-g in G-EAT is in marked contrast to the antifibrotic functionof IFN-g in SAT, as discussed below, and is also distinctfrom other inflammatory processes where the primary pro-fibrotic cytokines are IL-13 and transforming growth factor-b (TGF-b) (37–39).

Transgenic Mice

Transgenic mice have also provided useful tools to inves-tigate the function of specific cytokines in EAT. Transgenicexpression of IFN-g on TEC of the EAT resistant C57Bl=6strain resulted in thyroid abnormalities, hypothyroidism,and limited CD4þ T cell infiltration (40). When the IFN-gtransgene was expressed on thyrocytes of SAT- and EAT-susceptible NOD.H-2h4 mice, there was increased expressionof MHC class II on thyrocytes, but the mice did not developspontaneous thyroiditis (41). After immunization with MTg,IFN-g-transgenic NOD.H-2h4 mice developed less severe dis-ease and reduced IgG1 responses compared toWT littermates(41). These studies are consistent with a disease-limiting roleof IFN-g in experimentally induced models of autoimmunethyroiditis, and suggest that local IFN-g activity in the thy-roid is sufficient for disease suppression (20). IL-12, a cyto-kine that promotes IFN-g production, was also expressed as atransgene on thyrocytes (42). IL-12 transgenic mice also de-veloped hypothyroidism. Although they developed onlymildthyroiditis spontaneously, immunization with MTg and ad-juvant induced more severe lymphocytic thyroiditis thanin similarly immunized WT littermates (42). The disease-promoting effect of IL-12 in transgenic mice was shown to beindependent of IFN-g (42). IL-12 also promotes developmentof very severe EAT in nontransgenic mice (12,43,44). Thepresence of IL-12 during in vitro activation of sensitizedsplenocytes promotes activation of cells that transfer verysevere G-EAT, and neutralization of IL-12 in vitro inhibitsactivation of effector cells for G-EAT (12). However, IL-12,like IFN-g, can have different effects on development of EATor G-EAT depending on when it is administered or neu-tralized. For example, cells from IL-12-deficient donors sen-sitized with MTg and adjuvant can be activated to transfersevere EAT and G-EAT, and IL-12 can inhibit EAT andG-EAT when administered in vivo (43,44). Some effects of

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IL-12 are independent of IFN-g; that is, splenocytes fromIFN-g–=– mice transfer severe G-EAT after activation withMTg and IL-12 in vitro (22). The effects of IL-12 deficiency onEAT and G-EAT are complicated by the fact that the IL-12–=–

mice used in the studies mentioned above were IL-12 p40–=–

mice, which are now known to be deficient in both IL-12and IL-23 (45). To our knowledge, IL-12 p35–=– mice have notbeen tested for their ability to develop EAT or G-EAT, andthe role of the IL-17-producing subset of CD4þ T cells [whichwould be compromised by IL-23 deficiency (46)] has not yetbeen addressed in murine models of EAT or G-EAT.

These apparently contradictory results of the effects ofIFN-g in EAT and G-EAT may reflect actual opposing effectsof IFN-g in the pathogenesis of EAT; that is, IFN-g may haveeffects that are both disease promoting and disease resolvingin EAT and G-EAT. This would be consistent with the knownopposing and often paradoxical functions of IFN-g in manymodels of inflammation and autoimmunity (47). IFN-g canupregulate expression of MHC class I and MHC class IImolecules on thyrocytes (1,18,48,49), providing targets forrecognition of TEC by CD4þ T cells and CD8þ T cells. IFN-gcan also be proapoptotic and antiproliferative, and can in-crease damage to the thyroid by inducing apoptosis ofthyrocytes or inhibit inflammation by inducing apoptosis ofinfiltrating inflammatory cells (14,26–28,50,51). The balanceof these opposing deleterious and beneficial effects of IFN-g(or IL-12 through its ability to promote IFN-g production)may direct the progression of inflammation in EAT andG-EAT, as well as in other autoimmune diseases.

Role of IFN-c in SAT

The model of SAT in NOD.H-2h4mice, like the EATmodels discussed above, has some similarities to Hashimoto’sthyroiditis in humans, with chronic infiltration of the thyroidby mononuclear cells and circulating autoantibody to thyro-globulin (52–55). When NOD.H-2h4mice are given 0.05%sodium iodide (NaI) in their drinking water, thyroid lesionsdevelop in nearly 100%of mice of both sexes in 8weeks (52–54). Previous work by our group and by others have shownthat both CD4þ and CD8þ T cells as well as B cells are re-quired for development of SAT (53,54,56–58). Both Th1 andTh2 cytokines are expressed in thyroids of NOD.H-2h4micewith SAT (53,59), and cells producing IL-12 and IFN-g aredetected in NOD.H-2h4 thyroids during the early phase ofSAT development (59). In contrast to the ability of IFN-g toboth promote and suppress EAT and G-EAT in mice, IFN-g isessential for development of typical lymphocytic (L)-SAT inNOD.H-2h4mice. IFN-g–=– NOD.H-2h4mice do not developL-SAT (60,61), and L-SAT develops only in mice with thy-rocytes able to respond to IFN-g; that is, IFN-gR–=– NOD.H-2h4mice also do not develop L-SAT (62). Thus the roleof IFN-g in SAT is very different from its role in EAT orG-EAT. Indeed, L-SAT in WT NOD.H-2h4mice is one ofrelatively few spontaneous or experimentally induced auto-immune diseases in mice that is absolutely dependent onIFN-g; that is, IFN-g–=– mice develop many other autoim-mune diseases, including scleroderma, diabetes, experi-mental allergic encephalomyelitis (EAE), collagen-inducedarthritis, experimental autoimmune uveitis, and systemiclupus erythematosus (SLE), in addition to EAT and G-EATas discussed above.

IFN-g has a dual role in SAT. It is absolutely required fordevelopment of classical L-SAT, but IFN-g suppresses devel-opment of another autoimmune lesion in NOD.H-2h4 mice,characterized by abnormal TEC hyperplasia=proliferationand fibrosis (60–62). Our laboratory developed IFN-g–=– andIFN-gR–=– NOD.H-2h4mice (60–62). Although neither straindevelops L-SAT, all IFN-g–=– and IFN-gR–=– mice given NaIin their drinking water develop abnormal proliferation andhyperplasia of thyroid epithelial cells (TEC H=P). About 60–70%of IFN-g–=– NOD.H-2h4 mice develop very severe TECH=P, in which almost the entire thyroid consists of largemasses of proliferating thyrocytes. All thyroids with severeTEC H=P also have some infiltrating cells, primarily T cells,macrophages, and some eosinophils (61). The infiltratinglymphocytes are presumably required for development ofsevere TEC H=P because IFN-g–=– NOD.H-2h4 SCID mice donot develop TEC H=P (61). Although thyroids of IFN-g–=–

NOD.H-2h4mice with TEC H=P have few infiltrating B cellsor plasma cells (both of which are prevalent in thyroids ofWT mice with L-SAT), all mice with TEC H=P produce anti-Tg autoantibodies. All NOD.H-2h4mice with severe TECH=P have low serum T4, and there is extensive deposition ofcollagen (fibrosis) surrounding the clusters of proliferatingthyrocytes (61). Splenocytes from IFN-g–=– mice with severeTEC H=P transfer severe TEC H=P with accelerated kineticsto IFN-g–=– NOD.H-2h4 SCID mice, suggesting that TEC H=Phas an autoimmune basis (61). Therefore, a single cytokine,IFN-g, is absolutely essential for development of L-SAT andto inhibit thyrocyte hyperplasia in WT NOD.H-2h4mice. Inthe absence of IFN-g, mice are resistant to L-SAT but theydevelop TEC H=P, which also has an autoimmune basis.

Adoptive transfer of WT splenocytes (as a source of IFN-g)to IFN-g–=– mice results in typical L-SAT, and TEC H=P issuppressed (60,62). In IFN-g–=– mice given WT splenocytes,both donor (WT) and recipient (IFN-g–=–) T cells are present inthe thyroid infiltrates (62), indicating that cells from IFN-g–=–

mice are able to migrate to the thyroid if IFN-g is provided.Since cells in IFN-gR–=– mice cannot respond to IFN-g,IFN-gR–=– mice were used to distinguish the effects of IFN-gon lymphocytes versus thyrocytes. Transfer of WT spleno-cytes or bone marrow as a source of IFN-g to IFN-gR–=–

NOD.H-2h4mice did not result in L-SAT or inhibit TEC H=P,although the same pool of WT splenocytes or bone marrowinduced L-SAT and inhibited TEC H=P in IFN-g–=– recipients(62). These results indicate that thyrocytes must be able torespond to IFN-g for development of L-SAT and inhibition ofTEC H=P. Unexpectedly, IFN-gR–=– splenocytes do not in-duce L-SAT in IFN-g–=– mice even though IFN-gR–=– lym-phocytes produce as much IFN-g as lymphocytes from WTdonors. Further studies indicated that recipients of IFN-gR–=–

splenocytes or bone marrow express less mRNA for IFN-g-inducible chemokines compared to recipients of WT cells(62), and upregulation of IFN-g-inducible chemokines maybe required to induce optimal migration of lymphocytes tothyroids (29–33). These results suggest that lymphocytesmust be able to respond to IFN-g to be induced to migrate tothe thyroid in sufficient numbers to result in L-SAT, andthyrocytes must express IFN-gR for SAT to develop.

Thyrocytes responding to IFN-g produced locally bythyroid-infiltrating inflammatory cells upregulate MHC classII molecules (41,48,49), and locally produced IFN-g may alsofunction to induce expression of chemokines or adhesion

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molecules on thyrocytes that are needed for development ofL-SAT. In support of this idea, it has been shown that iodineand IFN-g act synergistically to increase ICAM-1 expressionon NOD.H-2h4 thyrocytes (63,64).

Role of IFN-c in Thyroid Fibrosis

Another example of the opposing functions of IFN-g inautoimmune thyroid diseases in mice concerns its role in fi-brosis in G-EAT versus SAT. As mentioned above, mice withvery severe G-EAT have extensive collagen deposition (fibro-sis) in their thyroids (13,14,25), and thyroids of IFN-g–=–

NOD.H-2h4mice with severe TEC H=P also have extensivefibrosis (61). In G-EAT, thyroid fibrosis occurs as a result of anexcessive proinflammatory response, in which there is a pre-ponderance of proinflammatory cytokines (including IFN-g),overproduction of the profibrotic cytokine TGF-b, and de-creased production of IL-10 in the thyroid (13,14,25,65). Fi-brosis in G-EAT is associated with increased apoptosis ofthyrocytes, and apoptosis of inflammatory cells is decreased(13,14). Neutralization of TGF-b inhibits fibrosis and promotesresolution of G-EAT (65). Although IFN-g–=– DBA=1 mice alsodevelop severe G-EAT, they have less fibrosis and increasedapoptosis of inflammatory cells resulting in earlier resolutionof lesions in comparison to their WT littermates (13,14). Thus,IFN-g tends to be profibrotic in G-EAT since fibrosis is in-creasedwhen IFN-g is highly expressed and is decreasedwhenIFN-g is absent. In the NOD.H-2h4 model, fibrosis is inhibitedby IFN-g, since WT NOD.H-2h4 mice that produce IFN-g anddevelop L-SAT never develop thyroid fibrosis, while NOD.H-2h4mice that develop severe TEC H=P due to lack of IFN-gor IFN-gR have extensive fibrosis (60–62). In addition, whenWT splenocytes (as a source of IFN-g) are transferred to IFN-g–=– NOD.H-2h4mice, fibrosis is inhibited (61). Fibrosis in IFN-g–=– NOD.H-2h4mice with TEC H=P is due, at least in part, tooverproduction of the profibrotic cytokine TGF-bby prolifer-ating thyrocytes. Transgenic mice expressing TGF-bon TECdevelop severe thyrocyte hyperplasia and extensive fibrosis,and neutralization of TGF-b inhibits development of severeTEC H=P and fibrosis in SCID recipients of IFN-g–=– spleno-cytes (Yu et al., manuscript in preparation). Therefore, IFN-g is primarily antifibrotic in SAT, as has also been observed inseveral other experimental models (37–39).

Role of IFN-c in Mouse Models of GD

Autoimmune GD, the most common endocrine disorderin humans, is mediated by autoantibodies that bind to thethyrotropin receptor (TSHR) and stimulate thyroid hormoneproduction (66). GD in humans is considered to be primarilya Th2-type disease with production of Th2 cytokines such asIL-4 and IL-10 generally predominating over Th1 cytokinessuch as IFN-g (67). There are a number of studies concerningthe role of Th1 versus Th2 cytokines in various murinemodels of GD. Although some studies suggest a role for bothIFN-g and IL-4 in some murine models of GD (68–70),knockout of IFN-g generally does not prevent developmentof GD, whereas knockout of IL-4 does inhibit disease de-velopment (69,71). Similarly, knockout of the transcriptionfactor STAT-6, which is needed for production of IL-4, in-hibited development of GD, whereas knockout of STAT-4,which is needed for production of IFN-g, resulted in a higherincidence and greater severity of hyperthyroidism (72).

However, IFN-g was shown to play an important role in amouse model of GD induced by TSHR antigen immuniza-tion (73). It is not clear which of the various animal models ofGD most closely mimics human GD, and multiple animalmodels must be analyzed to gain an insight into the patho-genesis of human GD (66).

Summary

Studies from murine models of autoimmune thyroiditisindicate that IFN-g is critical for the development of SAT inNOD.H-2h4mice, but it is not absolutely required for de-velopment of EAT, G-EAT, or GD in mice. In EAT, G-EAT,and GD, IFN-g can have augmenting or suppressive effectson disease development depending on various factors suchas the amount, site of expression, and the presence or absenceof IFN-g at various stages of the autoimmune inflammatoryresponse. The diverse and often opposing roles of IFN-gin autoimmune thyroid diseases are not unexpected, sinceparadoxical roles of IFN-g in other inflammatory and auto-immune diseases have been recognized by others (47). UsingIFN-g as an example of only one of many cytokines involvedin regulation of autoimmune diseases suggests that it is im-portant to exercise caution in making generalizations withregard to the role played by one specific cytokine in a givenautoimmune disease.

Acknowledgments

The authors thank Gordon Sharp for his helpful insights ininterpretation of studies from our laboratory cited in thisreview. The work from our laboratory was supported by theNIH Grant DK 35527 and by a VA Merit Review Grant.

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Address reprint requests to:Helen Braley-Mullen

Division of ImmunologyDepartment of MedicineUniversity of MissouriColumbia, MO 65212

E-mail: mullenh@health.missouri.edu

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