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. Author manuscript; available in PMC: 2012 Feb 4.
Published in final edited form as: J Immunol. 2010 Mar 24;184(9):5352–5359. doi: 10.4049/jimmunol.0903620

Transgenic expression of TGF-β on thyrocytes inhibits development of spontaneous autoimmune thyroiditis and increases regulatory T cells in thyroids of NOD.H-2h4 mice

Shiguang Yu *,^,§, Yujiang Fang *,^, Gordon C Sharp ^,#, Helen Braley-Mullen *,^,‡,2
PMCID: PMC3272275  NIHMSID: NIHMS352719  PMID: 20335535

Abstract

Transgenic NOD.H-2h4 mice expressing TGF-β under control of the thyroglobulin promoter were generated to address the role of TGF-β in development of thyrocyte hyperplasia. In contrast to non-transgenic (Tg−) littermates which develop lymphocytic spontaneous autoimmune thyroiditis (L-SAT), all TGF-β transgenic (Tg+) mice given NaI water for 2–7 mo develop thyroid lesions characterized by severe thyroid epithelial cell hyperplasia and proliferation (TEC H/P) with fibrosis and less lymphocyte infiltration than in nontransgenic mice. Most TGF-β transgenic mice produced less anti-mouse thyroglobulin (MTg) autoantibody than wild type (WT) mice. T cells from TGF-β transgenic and WT mice were equivalent in their ability to induce L-SAT after transfer to SCID or TCRα−/− mice. WT lymphocytes could transfer experimental autoimmune thyroiditis (EAT) or L-SAT to TGF-β transgenic mice, indicating that the transgenic environment did not prevent migration of lymphocytes to the thyroid. Thyroids of TGF-β transgenic mice had higher frequencies of Foxp3+ regulatory T cells (T reg) compared to nontransgenic WT mice. Transient depletion of T reg by anti-CD25 resulted in increased infiltration of inflammatory cells into thyroids of transgenic mice. T reg depletion also resulted in increased anti-MTg autoantibody responses and increased expression of IFN-γ and IFN-γ inducible chemokines in thyroids of TGF-β transgenic mice. The results suggest that SAT is inhibited in mice expressing transgenic TGF-β on thyrocytes, at least in part, because there is an increased frequency of T reg in their thyroids.

Keywords: Autoimmunity, inflammation, hyperplasia, TGF-β, T regulatory cells

Introduction

Several disease processes including some autoimmune diseases, as well as fibrosis and chronic inflammation, are characterized by dysregulated expression of or response to TGF-β (1). Effects of TGF-β on lymphocytes and other cells such as epithelial cells can be either stimulatory or inhibitory, depending on the cytokine milieu (2). For example, TGF-β inhibits T cell proliferation and promotes activation and function of regulatory T cells (T reg) (3, 4). TGF-β also influences the recruitment, adhesion and activation of circulating leukocytes (5), and can suppress production of pro-inflammatory cytokines by T cells and macrophages (2, 6). TGF-β can also be proinflammatory, e.g. in combination with IL-6 and antigen, TGF-β induces autoimmune inflammation mediated by Th17 cells (7–9).

NOD.H-2h4 mice develop lymphocytic spontaneous autoimmune thyroiditis (L-SAT)3 characterized by infiltration of the thyroid by B and T lymphocytes (10–15). IFN-γ is important for development of L-SAT, since IFN-γ−/− NOD.H-2h4 mice do not develop typical L-SAT, but instead develop thyroid epithelial cell hyperplasia and proliferation (TEC H/P) and fibrosis (16, 17). TEC H/P has an autoimmune basis, because lymphocytes are required for development of TEC H/P, and mice with TEC H/P produce anti-MTg autoantibodies (17). Splenocytes from IFN-γ−/− mice with severe TEC H/P transfer severe TEC H/P and fibrosis to NOD.H-2h4.SCID mice (17). Proliferating thyrocytes produce active TGF-β and anti-TGF-β inhibits TEC H/P and fibrosis in SCID recipients of IFN-γ−/− donor splenocytes with severe TEC H/P, indicating that TGF-β promotes development of TEC H/P and fibrosis in IFN-γ−/− NOD.H-2h4 mice (18).

In order to directly determine if TGF-β plays a role in development of TEC H/P and fibrosis, TGF-β transgenic (Tg) NOD.H-2h4 mice expressing active TGF-β on thyrocytes were generated. All TGF-β transgenic IFN-γ−/− NOD.H-2h4 mice developed moderate to severe TEC H/P and fibrosis, consistent with the role of TGF-β in promoting thyrocyte hyperplasia (18). Unexpectedly, expression of transgenic TGF-β on thyrocytes of WT NOD.H-2h4 mice inhibited development of L-SAT and transient depletion of T reg promoted development of typical L-SAT in TGF-β transgenic mice.

Materials and Methods

Generation of TGF-β transgenic mice

The TGF-β rat thyroglobulin promoter construct was provided by Dr. Leonard Kohn (Ohio University). The Sal I sites of the TGF-β rat thyroglobulin promoter construct was used to excise the cassette for microinjection. This construct contains two G to C point mutations in the TGF-β coding sequence that result in Cys to Ser amino acid substitutions at residues 223 and 225 of TGF-β1, resulting in a bioactive TGF-β1 (19–21). The construct was directly injected into superovulated NOD.H-2h4 mice (University of Missouri Transgenic Core), resulting in two founder TGF-β transgenic NOD.H-2h4 females. The founders were mated with NOD.H-2h4 males, and Tg+ offspring were selected by PCR analysis of tail DNA as previously described (16, 18). The TGF-β transgenic founders and progeny were genotyped by PCR analysis of mouse tail DNA using primers specific for the rat thyroglobulin promoter 5′-AGA GCA CTG CTT GCC ACT GTG C-3′ (forward), and 5′-GCT GTT GTA CAA AGC GAG CAC C-3′ (reverse) located in the mouse TGF-β genomic sequence and the transgenic vector. These primers amplify a 340bp band in mice expressing the TGF-β transgene.

Evaluation of TEC H/P and L-SAT severity scores

Male mice were used for all experiments. At various intervals after receiving NaI water, thyroids were removed, and one thyroid lobe from each mouse was fixed in formalin, sectioned and stained with hematoxylin and eosin (H & E). All slides were independently scored by at least two individuals, one of whom had no knowledge of the experimental groups. The other thyroid lobe was snap frozen in liquid nitrogen, and stored at −70°C for use in immunohistochemical staining or for isolation of RNA for RT-PCR. The extent of infiltration of thyroids by lymphocytes was scored on a scale of 0 to 4+ as previously described (11, 12). Briefly, a score of 0 indicates a normal thyroid or mild follicular changes with a few inflammatory cells (lymphocytes) infiltrating the thyroids. A 1+ severity score is defined as an infiltrate of at least 125 inflammatory cells in one or several foci, and 2+ represents 10–20 foci of inflammatory cell infiltration, each the size of several follicles causing replacement or destruction of up to 1/4 of the gland. A 3+ score indicates that 1/4 to 1/2 of the follicles are destroyed or replaced by infiltrating inflammatory cells and a 4+ score indicates that greater than 1//2 of the gland is destroyed. Because thyroids in all TGF-β transgenic mice had follicular hyperplasia and fibrosis which were never observed in thyroids of WT NOD.H-2h4 mice that did not express the transgene, the severity scores given in the Tables and Figures indicate the extent of infiltration of thyroids by inflammatory cells (primarily lymphocytes), i.e. L-SAT severity scores (11, 12).

Anti-mouse thyroglobulin (MTg) autoantibody determination

MTg-specific autoantibodies were determined by ELISA using serum from individual mice as previously described (11, 16, 17). Sera were diluted 1/50 or 1/100. Normal mouse serum used at the same dilutions always gave an OD value of <0.05. OD values >0.1 for experimental sera were considered positive.

Immmunohistochemical staining

TGF-β staining was done as described previously (18) using anti-TGF-β (AB-101-NA, R&D) to stain for active TGF-β (22), and chicken IgY as isotype control. Frozen thyroid sections were used for immunohistochemical staining as previously described (12, 16). The following primary antibodies were used: anti-CD4 (GK 1.5; ATCC), anti-CD8 (53.6; ATCC), anti-B220 (Invitrogen). Biotinylated goat anti-rat IgG (Invitrogen) was used as secondary antibody, and 0.3% hydrogen peroxide was used to block endogenous peroxidase. Sections were developed using the Vectastain Elite ABC kit (Vector Laboratories), and peroxidase activity was visualized using the Nova-Red substrate (Vector Laboratories). Slides were counterstained with hematoxylin. Negative controls used IgG isotype controls as primary antibody, with the remaining steps performed as described above. These controls were always negative.

Confocal laser scanning double-immunofluorescence microscopy

Foxp3 and CD4 double immunofluorescence staining was done using frozen sections of thyroids as previously described (18, 23). After blocking with 2% BSA, sections were incubated with anti-Foxp3 (rabbit anti-Foxp3, provided by Dr. Alexander Y. Rudensky (23)) 40 minutes at room temperature and visualized with 1:500 diluted Alexa 568 conjugated anti-rabbit antibody (Molecular Probes, Invitrogen). CD4 staining was visualized with 1:500 diluted Alexa 488 conjugated anti-rat IgG (Invitrogen). Slides were observed using a Bio-Rad Radiance 2000 confocal system coupled to an Olympus IX70 inverted microscope. The frequency of Foxp3+ cells among thyroid infiltrating CD4+ T cells was quantified by manually counting all cells in 2–3 randomly selected high power fields (magnification × 300), and Foxp3+ cells were expressed as a percentage of the total CD4+ T cells. Results represent the summary of data from 3 individual thyroids in each group and are expressed as the mean ± SE.

Semiquantitative and quantitative RT-PCR

Total RNA was isolated from thyroids or splenocytes using TRIZOL (Invitrogen), and cDNA was synthesized as previously described (12, 16). Semiquantitative RT-PCR was done as previously described (12, 16) using the housekeeping gene β-actin to correct for differences in the amount of RNA in different samples. Samples were electrophoresed, stained with ethidium bromide, and densitometry analysis was performed using a digital imaging system. Samples within the linear relationship between input cDNA and final PCR products (usually 1/25 dilution of cDNA) were collected, and densitometric units for each cytokine band were normalized to that for the corresponding β-actin band (16). Results are expressed as ratios of cytokine/β-actin. A ratio of 100 indicates a 1:1 ratio between a particular cytokine and β-actin. Quantitative RT-PCR was done using Absolute QPCR SYBR green ROX mix (ABgene) and ABI PRISM 7000 sequence detection system (Applied Biosystems, CA). A series of five standards with defined values were included in every reaction and a standard curve was obtained to calculate the amount of gene amplified. A dissociation curve was generated at the end of each PCR to verify the amplification of a single product. The level of β-actin expression for each sample was used for data normalization (23).

TGF-β ELISA

Active TGF-β in serum or in supernatants of thyrocyte cultures of TGF-β transgenic or nontransgenic mice was analyzed using a TGF-β ELISA kit (eBioscience). Samples were activated by treating with 1N HCl and neutralized with 1N NaOH according to the manufacturer’s instructions. Results from 4–5 samples in each group are expressed as the mean ± SE.

Flow cytometry

Spleen or cervical lymph node (CLN) cells from experimental mice were analyzed by flow cytometry (FACScan) as described previously (15). CD4-FITC, CD25-PE-Cy5, and Foxp3 T reg kits were obtained from eBioscience (23).

Adoptive transfer experiments

For some experiments, splenocytes (3 × 107 cells) from naive WT NOD.H-2h4 mice or from WT NOD.H-2h4 mice with SAT were transferred i.v. to TGF-β transgenic mice. Recipients were irradiated (300 rad) prior to cell transfer, recipients were given 0.05% NaI water, and thyroids were removed 8 wk later (11, 12). In other experiments, T cells from WT or TGF-β transgenic NOD.H-2h4 mice were obtained by passage of splenocytes through nylon wool (24). Enriched T cells were transferred i.v. to 300 rad irradiated TCR α−/− NOD.H-2h4 mice lacking detectable CD4+ and CD8+ T cells. Recipients were given NaI water and thyroids were removed 8 wk later.

Transfer of experimental autoimmune thyroiditis (EAT) to TGF-β transgenic mice

For some experiments, WT NOD.H-2h4 mice were immunized with MTg and lipopolysaccharide (LPS) as previously described (25). Splenocytes from immunized donors were activated in vitro with MTg and 5 ng/ml IL-12. Cells were transferred i.v. to 500R irradiated TGF-β transgenic or nontransgenic littermates, and thyroids were removed 21 days later (25).

Depletion of T reg

As previously described (23), some mice were given 3 weekly injections of 0.5 mg rat anti-mouse CD25 mAb PC61 (ATCC) or isotype control beginning 9–15 days after birth. At 8 wk of age, treated and control mice were given 0.05% NaI water and thyroids were removed 8 wk later.

Statistical Analysis

The Wilcoxon Rank Sum test was used for analyzing differences in disease severity scores between groups of mice, and the Students t test was used for all other analyses. A value of P < 0.05 was considered significant.

Results

Generation of transgenic NOD.H-2h4 mice expressing TGF-β on thyrocytes

The rat thyroglobulin promoter/active TGF-β1 construct was directly injected into superovulated NOD.H-2h4 mice to produce transgenic mice expressing TGF-β on thyrocytes. Transgenic founders were crossed with WT NOD.H-2h4 mice and the progeny were examined for expression of TGF-β by RT-PCR and immunohistochemical staining (Fig. 1). Quantitative analysis of TGF-β by real-time PCR indicated that thyroids of TGF-β transgenic NOD.H-2h4 mice expressed 3–5 fold more TGF-β than thyroids of nontransgenic mice (Fig. 1A). Immunohistochemical staining showed that thyrocytes of transgenic mice strongly expressed active TGF-β, whereas thyroids of nontransgenic littermates expressed little if any active TGF-β (Fig. 1C vs Fig. 1G). Salivary glands (Fig. 1D), spleen (Fig. 1E) and CLN (not shown) of transgenic mice expressed little or no TGF-β, indicating that the TGF-β transgene was expressed specifically on thyrocytes. The latent form of TGF-β was comparably expressed in thyroids of both transgenic and nontransgenic mice (data not shown).

Figure 1.

Figure 1

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Expression of TGF-β in NOD.H-2h4 mice expressing transgenic TGF-β on thyrocytes. (A). Real time PCR indicated that TGF-β mRNA expression in thyroids of transgenic mice was 3–5 fold higher than in nontransgenic WT mice. Thyroids of TGF-β transgenic mice express high levels of TGF-β (C) whereas salivary gland and spleen (D, E) express little or no TGF-β. TGF-β is not expressed by thyrocytes, salivary gland and spleen in naïve WT NOD.H-2h4 mice (G—I), TGF-β isotype control staining (B, F). Magnification: B–I, 400X.

Transgenic TGF-β expressed by thyrocytes is not detected in serum or supernatants of thyrocyte cultures

Others have reported that serum levels of TGF-β were increased in TGF-β transgenic mice (19), suggesting that transgenic TGF-β could potentially exert some of its functions peripherally. Serum levels of active TGF-β in individual serum samples from 6 TGF-β transgenic and 6 WT NOD.H-2h4 mice were determined by ELISA. Both transgenic and nontransgenic mice had similar low levels of serum TGF-β (698±170 pg/ml vs 571±104 pg/ml, P>0.5). In addition, supernatants of cultured thyrocytes from TGF-β transgenic and nontransgenic littermates produced similar amounts of active TGF-β (466±168 pg/ml vs 381±47 pg/ml, P>0.5) as determined by ELISA. These results suggest that transgenic TGF-β expressed by thyrocytes is not secreted, and are consistent with reports that serum levels of TGF-β1 in TGF-β transgenic NOD mice expressing TGF-β in pancreatic islets did not differ from those in nontransgenic littermates, although development of diabetes was inhibited by the transgenic TGF-β (26).

Transgenic expression of TGF-β on thyrocytes inhibits development of L-SAT in WT NOD.H-2h4 mice

SAT in WT NOD.H-2h4 mice is characterized by infiltration of thyroids by T and B lymphocytes, and almost all WT NOD.H-2h4 mice develop L-SAT 2 mo after receiving 0.05% NaI in their drinking water (11, 12). In contrast, all TGF-β transgenic (Tg+) NOD.H-2h4 mice developed moderate to severe thyroid epithelial cell hyperplasia and proliferation (TEC H/P) (Fig. 2A, B, asterisks) with extensive collagen deposition (fibrosis) (Fig. 2C) 2 mo after receiving NaI water, but they developed only minimal L-SAT (Fig. 2B, arrows) even after receiving NaI in their water for as long as 6–7 mo (Table I). Nontransgenic littermates, like WT NOD.H-2h4 mice, developed typical L-SAT with clusters of infiltrating lymphocytes (Fig. 2D, E, arrows), and they did not have TEC H/P or fibrosis (Table I, Fig. 2D, E, F). Thyroid lesions in TGF-β transgenic mice are similar to TEC H/P lesions that develop in IFN-γ−/− NOD.H-2h4 mice (17), except thyroids in the transgenic mice are smaller, TEC H/P develops much earlier and fibrosis is more extensive. Notably, most lymphocytes in thyroids of TGF-β transgenic mice accumulated near the periphery of the thyroid (data not shown), whereas in nontransgenic mice most lymphocytes infiltrated the gland (Fig. 2D, E, arrows). Thus, L-SAT severity is markedly reduced and TEC H/P and fibrosis is increased in transgenic mice overexpressing TGF-β on thyrocytes.

Figure 2.

Figure 2

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Representative thyroid histology from experiments in Table I and Table III. TGF-β transgenic mice develop severe thyrocyte hyperplasia (A, B, asterisks) and fibrosis (C) with minimal lymphocyte infiltration (arrows), whereas nontransgenic littermates develop L-SAT after 2 mo on NaI water (D, E, F, arrows). (G–L) Splenocytes from WT NOD.H-2h4 mice immunized with MTg and LPS were activated in vitro with MTg and IL-12 and transferred i.v. to 500R irradiated Tg+ (G, H, I) or Tg− recipients (J, K, L). Transgenic and nontransgenic recipients of the same donor splenocytes developed comparable G-EAT severity (Table III) and Trichrome staining showed that fibrosis was reduced in thyroids of transgenic recipients (I) compared to TGF-β transgenic mice that did not receive cells (C). Magnification: A, D, G and J, 100X; B, C, E, F, H, I, K, and L 400X. Panels C, F, I and L are Trichrome stains while the other panels are H&E stains.

Table I.

Development of L-SAT in Tg− and Tg+ NOD.H-2h4 mice given NaI water for 2-7 mo

L-SAT Severityb
Micea 0 1+ 2+ 3+ 4+ Anti-MTgcIgG
Tg− 2-3 mo NaI 1 1 5 4 3 0.698± 0.122
Tg+ 2-3 mo NaI 9 3 2 1 0 0.371± 0.069
Tg− 4-5 mo NaI 1 3 6 1 0 0.485±0.059
Tg+ 4-5 mo NaI 7 2 1 0 0 0.337±0.0563
Tg− 6-7 mo NaI 0 1 6 2 2 0.458±0.111
Tg+ 6-7 mo NaI 9 3 2 0 0 0.234±0.056
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a

7–8 wk old male TGF-β transgenic NOD.H-2h4 mice or nontransgenic littermates were given 0.05% NaI in their water at 8 wk of age, and thyroids were removed 2–7 mo later as indicated.

b

Numbers of mice with various degrees of severity of L-SAT. P<0.01 (line 1 vs line2, line 3 vs line 4, line 5 vs line 6);

c

Anti-MTg IgG (1/50 serum dilutions) expressed as OD410 ± SEM. P<0.05 (line 1 vs line 2); P< 0.1 (line 3 vs. line 4); P<0.05(line 5 vs line 6).

Anti-MTg autoantibody responses are reduced in transgenic mice expressing TGF-β on thyrocytes

All IFN-γ−/− mice with severe TEC H/P and fibrosis and WT NOD.H-2h4 mice with L-SAT produce anti-MTg autoantibodies, but autoantibody levels are generally higher in WT mice with L-SAT than in IFN-γ−/− mice with TEC H/P (17). Because TGF-β transgenic WT NOD.H-2h4 mice given NaI water for 2–7 mo have TEC H/P and fibrosis and minimal L-SAT (Table I), it was important to determine if anti-MTg autoantibody production was influenced in mice expressing TGF-β in the thyroid. Although there was considerable variation in anti-MTg autoantibodies in individual mice, TGF-β transgenic mice generally had lower levels of anti-MTg autoantibodies compared to nontransgenic mice (Table I). Since transgenic expression of TGF-β was confined to the thyroid, these results were unexpected, and suggest that the immune response to MTg might be influenced by expression of the transgene in the thyroid.

To determine if TGF-β transgenic mice could develop anti-MTg autoantibody responses comparable to those of WT mice after immunization with MTg and adjuvant, groups of age matched transgenic and nontransgenic mice were immunized with MTg and LPS and serum was collected 14 days later. Both groups produced comparable levels of anti-MTg autoantibody (data not shown), suggesting that expression of transgenic TGF-β in the thyroid did not influence the ability of B cells to respond to immunization with MTg and adjuvant.

T cells from transgenic and nontransgenic mice induce comparable L-SAT after transfer to TCR α−/− or SCID mice

To determine if autoreactive T cell activation was reduced in TGF-β transgenic mice, T cells were purified from spleens of transgenic and nontransgenic mice, and transferred to TCRα−/− NOD.H-2h4 mice. Recipients were given NaI water and thyroids were removed 2 mo later. TCRα−/− mice given T cells from either transgenic or nontransgenic mice developed comparable SAT, and both groups produced similar levels of anti-MTg autoantibody (Table II). The autoantibody responses and SAT development were dependent on the transferred T cells, since TCRα−/− mice that did not receive T cells did not develop SAT and did not produce detectable autoantibody (data not shown). These results suggest that when they are removed from the transgenic environment, autoreactive T cells in TGF-β transgenic mice are as effective as those from nontransgenic mice in terms of their ability to induce SAT. In other experiments, T cells from WT and TGF-β transgenic mice also induced comparable L-SAT when transferred to SCID recipients (data not shown).

Table II.

Development of L-SAT in TCRα−/− recipients of splenocytes from Tg− or Tg+ Donors

L-SAT Severityb
Cell transfera 0 1+ 2+ 3+ Anti-MTg IgGc
Naïve Tg− 0 3 3 0 0.574 ± 0.125
Naïve Tg+ 0 4 2 0 0.469 ± 0.104
Naive Tg− 1 3 1 0 0.239 ± 0.047
Naive Tg+ 1 3 2 0 0.244 ± 0.055
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a

Splenic T cells (107) from naive Tg− and Tg+ mice (8–10 wk old) were transferred to 300 rad irradiated TCRα−/− mice. Mice were given NaI in their drinking water, and thyroids were removed 2 mo later for evaluation of thyroid histopathology.

b

P=0.64 (line 1 vs line 2), and P=0.45 (line 3 vs line 4).

c

see Table I.

Lymphocytes from WT mice infiltrate thyroids to induce G-EAT and L-SAT after transfer to TGF-β transgenic recipients

The results in Fig. 2 and Table I indicate that lymphocyte infiltration of the thyroid is reduced in most mice expressing transgenic TGF-β on thyrocytes. To determine if the transgenic TGF-β prevented migration of lymphocytes to thyroids of transgenic mice, WT NOD.H-2h4 mice were immunized with MTg and LPS, and their splenocytes were activated with MTg and IL-12 in vitro prior to transfer to transgenic or WT recipients. TGF-β transgenic and nontransgenic recipients of MTg-sensitized and activated splenocytes developed severe granulomatous EAT (G-EAT) characterized by infiltration of thyroid by CD4+ and CD8+ T cells, plasma cells, and macrophages (Table III, Fig. 2G, H vs. J, K) and both groups had similar anti-MTg responses (Table III). These results indicate that lymphocytes activated in vitro in the presence of IL-12 can traffic normally to thyroids that overexpress TGF-β. Interestingly, fibrosis in thyroids of transgenic recipients of MTg-sensitized and activated Tg− splenocytes was reduced compared to that of transgenic mice not receiving activated splenocytes (Fig. 2I vs. C). Splenocytes from MTg and LPS immunized WT and TGF-β transgenic donors activated in vitro with MTg and IL-12 also transferred equivalent G-EAT to WT NOD.H-2h4 recipients (data not shown).

Table III.

Tg− and Tg+ NOD.H-2h4 mice recipients of WT splenocytes develop comparable G-EAT

EAT Severityb
Recipientsa 0 1+ 2+ 3+ 4+ Anti-MTgc IgG
Tg− 0 0 0 1 4 0.784 ± 0.064
Tg+ 0 0 1 3 0 1.258 ± 0.197
Tg− 0 0 0 1 4 0.778 ± 0.220
Tg+ 0 0 0 0 6 0.803 ± 0.206
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a

NOD.H-2h4 mice were immunized with MTg and LPS as described in Methods. Splenocytes from immunized donors were activated in vitro with MTg and IL-12, and transferred i.v. to irradiated Tg− or Tg+ recipients. Thyroids were removed 21 days later for assessment of thyroid infiltration.

b

See Table I.

c

See Table I.

Splenocytes from naïve nontransgenic NOD.H-2h4 mice or from NOD.H-2h4 mice with L-SAT were also transferred to TGF-β transgenic mice. Lymphocyte infiltration of thyroids of TGF-β transgenic mice was increased following transfer of naïve splenocytes or splenocytes from WT mice with L-SAT, and anti-MTg autoantibody responses were higher than in transgenic mice not given WT lymphocytes (Table IV). Although transgenic recipients of WT lymphocytes still had TEC H/P, and fibrosis was not inhibited (data not shown), the results indicate that transgenic expression of TGF-β in thyroids did not prevent lymphocyte migration to thyroids.

Table IV.

Development of L-SAT in Tg+ recipients of naive or SAT NOD.H-2h4 splenocytes

L-SAT Severityb
Cell transfera 0 1+ 2+ 3+ Anti-MTg IgGc
None 5 0 0 0 0.129 ± 0.011
naive spleen 4 2 3 0 0.381± 0.064
L-SAT spleen 1 5 5 0 0.556± 0.086
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a

Splenocytes from naive WT NOD.H-2h4 mice (line 2) or from WT mice with 2-3+ L-SAT (line 3) were transferred to 300 rad irradiated TGF-β transgenic (Tg+) mice. Tg+ mice in line 1 received no cells. Mice were given NaI in their drinking water, and thyroids were removed 2 mo later for evaluation of thyroid histopathology.

b

See Table I. P =0.24 (line 2 vs line 3).

c

See Table I. P=0.134(line 2 vs line 3).

The frequency of Foxp3+ T reg cells is increased in thyroids of TGF-β transgenic mice

TGF-β plays a role in controlling the activation and function of CD4+CD25+Foxp3+ T reg. TGF-β can convert naïve CD4+CD25-Foxp3− T cells into CD4+CD25+Foxp3+ T reg (27), and it plays an important role in suppression of immune responses mediated by T reg (4). To determine if resistance to L-SAT in TGF-β transgenic mice might be explained, at least in part, by increased numbers and/or activity of T reg, Foxp3+ cells in thyroids, spleens and CLN of transgenic and nontransgenic mice were evaluated by immunohistochemical staining. The frequency of Foxp3+ T cells among total intrathyroidal CD4+ T cells was significantly increased in thyroids of TGF-β transgenic mice compared to nontransgenic littermates (Fig. 3A–D; p< 0.05). However, the total number of Foxp3+ cells in the thyroid was not increased because there were fewer infiltrating CD4+ T cells in thyroids of TGF-β transgenic mice compared to their transgene-negative littermates. The frequency of CD4+CD25+Foxp3+ cells was marginally increased in CLN of transgenic compared to nontransgenic mice, but there was no difference in the frequency of CD4+Foxp3+ cells in spleens of the two groups of mice (Fig. 3E). Thyroids of young (4 wk) transgenic or nontransgenic mice with no thyroid inflammation did not have detectable Foxp3+ cells, but CLN of young transgenic mice, similar to the older mice in Fig. 3E, had an increased frequency of Foxp3+ T cells compared to their nontransgenic littermates (Fig. 3F).

Figure 3.

Figure 3

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Analysis of Foxp3+CD4+ T cells in thyroids, spleen, and CLN of TGF-β transgenic and nontransgenic littermates. Foxp3+ (red) CD4+ (green) T cells were detected by confocal microscopy. Representative thyroid sections of transgenic mice (A, B) and nontransgenic littermates with 2-3+ L-SAT (C) are shown. D represents a summary of the percentage of Foxp3+CD4+ cells in intrathyroidal CD4+ T cells in three individual thyroids of transgenic vs. nontransgenic mice quantitated as described in Methods. Results in E represent the percentage of Foxp3+ CD4+ T cells in spleen and CLN of the same mice as determined by flow cytometry, and F represents the percentage of Foxp3+CD4+ T cells in CLN of young (4 wk old) transgenic and nontransgenic mice not given NaI water.

Transient depletion of T reg increases L-SAT and anti-MTg autoantibody responses in TGF-β transgenic mice

To determine if the increased frequency of Foxp3+ cells in thyroids of TGF-β transgenic mice might play a role in suppression of L-SAT, groups of transgenic and nontransgenic mice were given rat IgG or anti-CD25 to transiently deplete T reg as previously described (23). Mice were given NaI water at 8 wk, and thyroids removed 8 wk later. Compared to TGF-β transgenic mice that received rat IgG (Table V, Fig. 4A–E), thyroids of TGF-β transgenic mice given anti-CD25 had greatly increased lymphocyte infiltration, although they also had proliferating thyrocytes and fibrosis (Table V, Fig. 4F–J). MTg autoantibodies in T reg-depleted transgenic as well as nontransgenic mice were always higher than those in mice given rat IgG (p<0.01). Consistent with our previous studies (23), transient depletion of CD25+ T cells had little effect on L-SAT severity in nontransgenic WT NOD.H-2h4 mice (Table V). Thyroids of T reg depleted TGF-β transgenic mice had clusters of CD4+ T cells and B cells (Fig. 4I, J), similar to that seen in nontransgenic NOD.H-2h4 mice (Fig. 2D, E). These results suggest that development of L-SAT is inhibited in mice overexpressing TGF-β on thyrocytes, at least in part, because they have an increased frequency of T reg in their thyroids and CLN.

Table V.

SAT severity is increased in Tg+ NOD.H-2h4 mice given anti-CD25

SAT Severityb
Micea 0 1+ 2+ 3+ Anti-MTgcIgG
Tg+/rat-Ig 6 2 0 0 0.144 ± 0.059
Tg+/anti-CD25 0 0 4 10 0.536±0.079
Tg−/rat-Ig 4 4 4 2 0.246± 0.069
Tg−/anti-CD25 0 1 5 4 0.617± 0.111
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a

Tg+ NOD.H-2h4 mice and Tg− littermates were given 3 weekly injections of 0.5 mg anti-CD25(PC61) or rat Ig beginning 9–15 days after birth (23). At 8 wk of age, all mice were given 0.05% NaI water and thyroids were removed 6–8 wk later.

b

See Table I. P<0.01(line 1 vs line 2); P=0.11 (line 2 vs line 4); P=0.02 (line 3 vs line 4).

c

See Table I. P<0.01(line 1 vs line 2, line 3 vs line 4); P=0.551 (line 2 vs line 4).

Figure 4.

Figure 4

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Transient depletion of T reg leads to increased lymphocyte infiltration of thyroids in TGF-β transgenic mice. Representative thyroid sections of transgenic mice given rat IgG (A–E) or anti-CD25 (F–J) are shown. Thyroids of mice given anti-CD25 had increased lymphocyte infiltration (F, G) compared to controls given rat IgG (A, B). However, TEC H/P (G) and fibrosis (H) in transgenic mice given anti-CD25 was similar to that in rat IgG controls (B, C). Thyroids of TGF-β transgenic mice given anti-CD25 had clusters of CD4+ T cells (I) and B cells (J) typically seen in L-SAT. These were not present in thyroids of transgenic mice given rat IgG (D, CD4+ T cells, E, Bcells).

T reg depletion increases expression of IFN-γ and IFN-γ inducible chemokines in thyroids of TGF-β transgenic mice

Most TGF-β transgenic mice develop TEC H/P with minimal lymphocyte infiltration of thyroids. As shown above, transient depletion of T reg results in increased migration of lymphocytes to thyroids of TGF-β transgenic mice. To determine if T reg depletion resulted in increased expression of cytokines or chemokines in thyroids of TGF-β transgenic mice, RNA was isolated from thyroids of WT and TGF-β transgenic mice given rat Ig or anti-CD25. As expected, expression of TGF-β in thyroids of transgenic mice was not influenced by T reg depletion. Thyroids of both groups of transgenic mice expressed comparable amounts of TGF-β mRNA, and this was always greater than in thyroids of nontransgenic littermates (Fig. 5). Consistent with the decreased infiltration of T cells, thyroids of rat Ig treated TGF-β transgenic mice expressed less IFN-γ than thyroids of nontransgenic littermates with L-SAT (Fig. 5). Consistent with the increased T cell infiltration, IFN-γ expression in thyroids of T reg depleted TGF-β transgenic mice was increased, but T reg depletion had no effect on SAT severity or expression of IFN-γ in thyroids of nontransgenic littermates. Thyroids of TGF-β transgenic mice with minimal lymphocyte infiltration always had lower expression of IFN-γ inducible chemokines CXCL9, CXCL10, and CCL5, compared to WT NOD.H-2h4 mice with 2-3+ L-SAT, and expression of these chemokines was increased in thyroids of transgenic mice after depletion of T reg (Fig. 5). However, expression of several other chemokines such as CCR7 was similar in thyroids of all mice. These results indicate that T reg depletion promotes infiltration of T cells and expression of IFN-γ and IFN-γ inducible chemokines in thyroids even when there is high expression of transgenic TGF-β in the thyroid. Therefore when T reg are depleted, peripheral T cells apparently reach a threshold level of activation that allows them to traffic more effectively to the thyroid.

Figure 5.

Figure 5

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Expression of cytokine and chemokine mRNA in thyroids of transgenic or nontransgenic mice with or without T reg depletion. RNA was isolated from thyroids of the mice shown in Table IV. Thyroids of TGF-β transgenic mice express more TGF-β than thyroids of nontransgenic mice, and TGF-β mRNA levels are not affected by T reg depletion. Expression of IFN-γ and IFN-γ inducible chemokines, e.g. CXCL9, CCL5 and CXCL10, were lower in thyroids of TGF-β transgenic mice compared to nontransgenic mice with L-SAT. Expression of these molecules was increased in transgenic mice after T reg depletion and was comparable to that of nontransgenic mice. Expression of IFN-γ and IFN-γ inducible chemokines was unaffected by T reg depletion in nontrangenic mice. Other molecules, e.g. CCR7, were expressed at similar levels in all groups. Results represent mean ± SEM of 5 individual thyroids for each group.

Discussion

The results of this study demonstrate that transgenic expression of TGF-β on thyrocytes inhibits development of L-SAT in WT NOD.H-2h4 mice, and all TGF-β transgenic mice develop thyroid epithelial cell hyperplasia and fibrosis. These results are consistent with our earlier studies indicating that overexpression of TGF-β by thyrocytes of IFN-γ−/− mice correlates with the severity of TEC H/P and neutralization of TGF-β inhibits development of TEC H/P and fibrosis (18). Of particular interest, transient depletion of T reg in mice expressing TGF-β in the thyroid resulted in higher SAT severity scores, with increased infiltration of CD4+ T cells and B cells into thyroids (Table V, Fig. 4). However, transient depletion of T reg does not reduce fibrosis in thyroids of TGF-β transgenic mice.

Since TGF-β has multiple functions, overexpression of TGF-β by thyrocytes could inhibit development of L-SAT by multiple mechanisms. IFN-γ inducible chemokines are important for recruiting inflammatory cells to the site of inflammation (29–31), and TGF-β interferes with the functions of IFN-γ (28, 32). TGF-β mRNA expression was inversely correlated with expression of IFN-γ and IFN-γ inducible chemokines such as CXCL9, CXCL10, and CCL5 in thyroids of transgenic mice (Fig. 5). Therefore, one mechanism by which TGF-β inhibits development of L-SAT is to inhibit upregulation of IFN-γ and IFN-γ inducible chemokines in the thyroid resulting in migration of fewer lymphocytes to thyroids.

Because expression of the TGF-β transgene was confined to the thyroid (Fig. 1) and there was no evidence that the active TGF-β was secreted by the thyrocytes and present in the circulation, it was surprising that anti-MTg autoantibody responses were consistently reduced in TGF-β transgenic mice compared to their nontransgenic littermates (e.g. Table I). Although the reason for these results is not understood, it is possible the increased frequency of T reg in the TGF-β transgenic mice inhibited expansion of the autoreactive B cells in responses to the thyroid expressed autoantigen as shown recently in another model (33). This hypothesis is consistent with the observation that anti-MTg autoantibody responses were always higher after T reg depletion (Table V).

Mice with transgenic expression of TGF-β in pancreatic islets have been described by others (26, 34, 35). NOD mice expressing transgenic TGF-β in pancreatic beta cells were relatively resistant to passive transfer of diabetes with diabetogenic T cells compared to their nontransgenic littermates (26, 35). In our studies, activated autoreactive T cells from TGF-β transgenic and nontransgenic mice were equivalent in their ability to induce L-SAT after transfer to SCID (data not shown) or TCRα−/− mice (Table II). In addition, adoptively transferred lymphocytes from nontransgenic WT mice could migrate to and infiltrate thyroids of TGF-β transgenic mice (Table IV), and splenocytes from WT mice could be activated in vitro to transfer severe G-EAT to TGF-β transgenic mice (Table III). TGF-β transgenic recipients of activated G-EAT effector cells also had less thyrocyte hyperplasia (Fig. 2G, H) and fibrosis (Fig. 2I). These results indicate that transferred T cells can migrate to thyroids of transgenic mice expressing TGF-β on thyrocytes.

TGF-β can promote apoptosis of T cells (35, 36). Transgenic expression of TGF-β in pancreatic islets was shown to increase apoptosis of islet infiltrating T cells, suggesting this could be one mechanism by which TGF-β prevents diabetes (35). In the current study, numbers of TUNEL-+ apoptotic cells in thyroids of WT nontransgenic mice with 2-3+ SAT were very low, and did not differ from those detected in thyroids of TGF-β transgenic mice with severe TEC H/P and fibrosis (data not shown). Therefore, there is no evidence to suggest that transgenic TGF-β expressed in the thyroid prevents development of L-SAT by inducing apoptosis of thyroid infiltrating inflammatory cells.

Production of TGF-β by T reg is important for inhibition of immune responses and for expansion of T reg in vivo (4, 37), and TGF-β can promote conversion of peripheral CD4+CD25-Foxp3− T cells into Foxp3-expressing CD4+CD25+ T reg (27). Although the frequency of CD4+CD25+ Foxp3+ T cells in spleens of TGF-β transgenic mice did not differ from that in WT nontransgenic mice, the frequency of Foxp3+ T cells among total thyroid infiltrating CD4+ T cells was increased in thyroids of TGF-β transgenic mice (Fig. 3A, D). Increased frequency or activity of T reg apparently plays an important role in suppression of SAT in TGF-β transgenic mice, since transient depletion of T reg by administration of anti-CD25 resulted in markedly increased infiltration of lymphocytes into thyroids (Fig. 4F, G, I, J). Anti-MTg autoantibodies and expression of IFN-γ and IFN-γ inducible chemokines were also increased after T reg depletion (Table V, Fig. 5). T reg depletion did not reverse all of the thyroid abnormalities in TGF-β transgenic mice as their thyroids still had TEC H/P and fibrosis which were absent in thyroids of nontransgenic littermates. It is not entirely clear why transient T reg depletion had such dramatic effects in promoting lymphocyte migration to thyroids of TGF-β transgenic mice since activation of autoreactive T cells in the spleen was not reduced in TGF-β transgenic mice compared to their nontransgenic littermates (Table II) and activation of T cells able to induce L-SAT in TCRα −/− mice was not higher after T reg depletion (data not shown). The same transient T reg depletion had essentially no effect on L-SAT severity or expression of IFN-γ or IFN-γ inducible chemokines in nontransgenic WT mice (Table V, Fig. 5). It is possible that T reg depletion in TGF-β transgenic mice lowered the threshold of activation of autoreactive T cells as suggested in other studies (38). Anti-MTg autoantibody responses were consistently increased in both transgenic and nontransgenic mice after T reg depletion (Table V). Although increased autoantibody responses in TGF-β transgenic mice after T reg depletion are consistent with their increased severity of L-SAT, autoantibody responses were also increased after T reg depletion in nontransgenic littermates in which L-SAT severity and production of IFN-γ or IFN-γ inducible chemokines in the thyroid was not affected. The reason that transient T reg depletion consistently resulted in increased autoantibody responses in both transgenic and nontransgenic mice is unknown. Taken together, the results presented here suggest that transgenic overexpression of TGF-β on thyrocytes inhibits development of L-SAT in NOD.H-2h4 mice and this is due, at least in part, to an increased frequency and/or activity of T reg in thyroids of TGF-β transgenic mice.

Acknowledgments

Supported by a Merit Review Grant from the Department of Veterans Affairs, by NIH Grant R56/RO1 AI074857, the A. P. Green Foundation, the University of Missouri Research Council and University of Missouri Research Board.

We thank Edward Downey, Alicia Duren and Daniel Schnurr for technical assistance and the transgenic animal core facility at the University of Missouri for generating the TGF-β transgenic mice. We thank Dr. Dr. Leonard Kohn (Ohio University) for providing the rat-thyroglobulin promoter-linked TGF-β construct and Dr. Alexander Y. Rudensky (Memorial Sloan-Kettering Cancer Center) for providing the anti-Foxp3.

Footnotes

3

Abbreviations: SAT, spontaneous autoimmune thyroiditis; H & E., hematoxylin and eosin; MTg, mouse thyroglobulin; RTg, rat thyroglobulin; TEC H/P, thyroid epithelial cell (thyrocyte) hyperplasia and proliferation.

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