New Murine Model of Early Onset Autoimmune Thyroid Disease/Hypothyroidism and Autoimmune Exocrinopathy of the Salivary Gland.

Sixty to seventy percent of IFN-γ(-/-) NOD.H-2h4 mice given sodium iodide (NaI)-supplemented water develop a slow onset autoimmune thyroid disease, characterized by thyrocyte epithelial cell (TEC) hyperplasia and proliferation (H/P). TEC H/P develops much earlier in CD28(-/-) mice and nearly 100% (both sexes) have severe TEC H/P at 4 mo of age. Without NaI supplementation, 50% of 5- to 6-mo-old CD28(-/-)IFN-γ(-/-) mice develop severe TEC H/P, and 2-3 wk of NaI is sufficient for optimal development of severe TEC H/P. Mice with severe TEC H/P are hypothyroid, and normalization of serum thyroxine levels does not reduce TEC H/P. Activated CD4(+) T cells are sufficient to transfer TEC H/P to SCID recipients. Thyroids of mice with TEC H/P have infiltrating T cells and expanded numbers of proliferating thyrocytes that highly express CD40. CD40 facilitates, but is not required for, development of severe TEC H/P, as CD40(-/-)IFN-γ(-/-)CD28(-/-) mice develop severe TEC H/P. Accelerated development of TEC H/P in IFN-γ(-/-)CD28(-/-) mice is a result of reduced regulatory T cell (Treg) numbers, as CD28(-/-) mice have significantly fewer Tregs, and transfer of CD28(+) Tregs inhibits TEC H/P. Essentially all female IFN-γ(-/-)CD28(-/-) NOD.H-2h4 mice have substantial lymphocytic infiltration of salivary glands and reduced salivary flow by 6 mo of age, thereby providing an excellent new model of autoimmune exocrinopathy of the salivary gland. This is one of very few models where autoimmune thyroid disease and hypothyroidism develop in most mice by 4 mo of age. This model will be useful for studying the effects of hypothyroidism on multiple organ systems.

Mechanisms and kinetics of proliferation and fibrosis development in a mouse model of thyrocyte hyperplasia.

IFN-γ(-/-) NOD.H-2h4 mice develop autoimmune disease with extensive hyperplasia and proliferation of thyroid epithelial cells (TEC H/P) and fibrosis. Splenic T cells from donors with severe TEC H/P transfer TEC H/P to SCID recipients. The goal of this study was to determine what factors control TEC H/P development/progression by examining T cells, markers of apoptosis, senescence and proliferation in thyroids of SCID recipients over time. At 28days, T cell infiltration was maximal, thyrocytes were proliferating, and fibrosis was moderate. At days 60 and 90, thyroids were larger with more fibrosis. T cells, cytokines and thyrocyte proliferation decreased, and cell cycle inhibitor proteins, and anti-apoptotic molecules increased. T cells and thyrocytes had foci of phosphorylated histone protein H2A.X, indicative of cellular senescence, when TEC H/P progressed and thyrocyte proliferation declined. Some thyrocytes were regenerating at day 90, with irregularly shaped empty follicles and ciliated epithelium. Proliferating thyrocytes were thyroid transcription factor (TTF1)-positive, suggesting they derived from epithelial cells and not brachial cleft remnants.

Follicular B cells in thyroids of mice with spontaneous autoimmune thyroiditis contribute to disease pathogenesis and are targets of anti-CD20 antibody therapy.

B cells are required for development of spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice where they function as important APCs for activation of CD4(+) T cells. Depletion of B cells using anti-CD20 effectively inhibits SAT development. The goals of this study were to characterize the B cells that migrate to thyroids in SAT, and to determine whether anti-CD20 effectively targets those B cells in mice with established SAT. The results showed that most thyroid-infiltrating B cells in mice with SAT are follicular (FO) B cells. Expression of CD80, CD86, and CD40 was significantly increased on FO, but not marginal zone, splenic B cells after SAT development. Thyroid-infiltrating and peripheral blood B cells had lower expresion of CD20 and CD24 compared with splenic and lymph node FO B cells. Despite reduced CD20 expression, anti-CD20 depleted most B cells in thyroids of mice with established SAT within 3 d. B cell depletion in thyroids of mice given anti-CD20 was more complete and longer lasting than in spleen and lymph nodes and was comparable to that in blood. Circulation of B cells was required for effective and rapid removal of B cells in thyroids because preventing lymphocyte egress by administration of FTY720 abrogated the effects of anti-CD20 on thyroid B cells. Therefore, the FO subset of B cells preferentially contributes to SAT development and persistence, and anti-CD20 targeting of FO B cells effectively eliminates B cells in the target organ even though thyroid B cells have decreased CD20 expression.

Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers.

NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) with chronic inflammation of thyroids by T and B cells. B-cell deficient (B(-/-) ) mice are resistant to SAT but develop SAT if regulatory T (Treg) cells are transiently depleted. We established a transfer model using splenocytes from CD28(-/-)  B(-/-) mice (effector cells and antigen-presenting cells) cultured with or without sorted Treg cells from Foxp3-GFP wild-type (WT) or B(-/-) mice. After transfer to mice lacking T cells, mice given Treg cells from B(-/-) mice had significantly lower SAT severity scores than mice given Treg cells from WT mice, indicating that Treg cells in B(-/-) mice are more effective suppressors of SAT than Treg cells in WT mice. Treg cells from B(-/-) mice differ from WT Treg cells in expression of CD27, tumour necrosis factor receptor (TNFR) II p75, and glucocorticoid-induced TNFR-related protein (GITR). After transient depletion using anti-CD25 or diphtheria toxin, the repopulating Treg cells in B(-/-) mice lack suppressor function, and expression of CD27, GITR and p75 is like that of WT Treg cells. If B(-/-) Treg cells develop with B cells in bone marrow chimeras, their phenotype is like that of WT Treg cells. Addition of B cells to cultures of B(-/-) Treg and T effector cells abrogates their suppressive function and their phenotype is like that of WT Treg cells. These results establish for the first time that Treg cells in WT and B(-/-) mice differ both functionally and in expression of particular cell surface markers. Both properties are altered after transient depletion and repopulation of B(-/-) Treg cells, and by the presence of B cells during Treg cell development or during interaction with effector T cells.

Agonistic anti-CD40 induces thyrocyte proliferation and promotes thyroid autoimmunity by increasing CD40 expression on thyroid epithelial cells.

CD40 is expressed on cells of the immune system and in some tissues that are targets for autoimmune-mediated damage. It is not known if CD40 expression in target tissues plays a role in the pathology of autoimmune diseases. This study shows that agonistic anti-CD40 induces strong and sustained proliferation of thyroid epithelial cells (TECs), or thyrocytes, in IFN-γ(-/-) autoimmune-prone NOD and NOD.H-2h4 mice. TEC proliferation is accompanied by greatly increased expression of CD40 on TECs, development of fibrosis and hypothyroidism, and increased expression of proinflammatory molecules in thyroids. Bone marrow chimera experiments indicate that TEC expression of CD40 is required for anti-CD40-induced TEC proliferation, but lymphoid cells do not have to express CD40. TEC proliferation is reduced in wild-type mice given anti-CD40, presumably because they produce IFN-γ, which inhibits TEC proliferation. CD40 also increases on TECs during development of an autoimmune thyroid disease characterized by TEC hyperproliferation that develops spontaneously in IFN-γ(-/-) NOD.H-2h4 mice. TEC hyperproliferation development is accelerated in mice given agonistic anti-CD40. These studies provide new information regarding the role of target tissue expression of CD40 in development of autoimmunity and suggest that use of agonistic anti-CD40 for tumor therapy could result in autoimmune disease.

Reduced effectiveness of CD4+Foxp3+ regulatory T cells in CD28-deficient NOD.H-2h4 mice leads to increased severity of spontaneous autoimmune thyroiditis.

NOD.H-2h4 mice given NaI in their drinking water develop iodine-accelerated spontaneous autoimmune thyroiditis (ISAT) with chronic inflammation of the thyroid by T and B cells and production of anti-mouse thyroglobulin (MTg) autoantibody. CD28(-/-) NOD.H-2h4 mice, which have reduced numbers of CD4(+)Foxp3(+) regulatory T cells (Tregs), were developed to examine the role of Tregs in ISAT development. CD28(-/-) NOD.H2-h4 mice develop more severe ISAT than do wild-type (WT) mice, with collagen deposition (fibrosis) and low serum T4. CD28(-/-) mice have increased expression of proinflammatory cytokines IFN-γ and IL-6, consistent with increased mononuclear cell infiltration and tissue destruction in thyroids. Importantly, transferring purified CD4(+)Foxp3(+) Tregs from WT mice reduces ISAT severity in CD28(-/-) mice without increasing the total number of Tregs, suggesting that endogenous Tregs in CD28(-/-) mice are functionally ineffective. Endogenous CD28(-/-) Tregs have reduced surface expression of CD27, TNFR2 p75, and glucocorticoid-induced TNFR-related protein compared with transferred CD28(+/+) Tregs. Although anti-MTg autoantibody levels generally correlate with ISAT severity scores in WT mice, CD28(-/-) mice have lower anti-MTg autoantibody responses than do WT mice. The percentages of follicular B cells are decreased and those of marginal zone B cells are increased in spleens of CD28(-/-) mice, and they have fewer thyroid-infiltrating B cells than do WT mice. This suggests that CD28 deficiency has direct and indirect effects on the B cell compartment. B cell-deficient (B(-/-)) NOD.H-2h4 mice are resistant to ISAT, but CD28(-/-)B(-/-) mice develop ISAT comparable to WT mice and have reduced numbers of Tregs compared with WT B(-/-) mice.

Transient depletion of CD4+ CD25+ regulatory T cells results in multiple autoimmune diseases in wild-type and B-cell-deficient NOD mice.

Approximately 80% of female wild-type non-obese diabetic (WT NOD) mice spontaneously develop diabetes, whereas B-cell-deficient (B(-/-)) NOD mice are resistant to diabetes. B(-/-) mice are also resistant to other spontaneous and experimentally induced autoimmune diseases, including arthritis, systemic lupus erythematosus, Sjögren syndrome and thyroiditis. Under normal conditions, activation of self-reactive T cells in the periphery is limited by CD4(+) CD25(+) natural regulatory T (Treg) cells. B(-/-) NOD.H-2h4 mice, normally resistant to spontaneous autoimmune thyroiditis (SAT), develop SAT when Treg cells are depleted, suggesting that Treg cells are preferentially activated when autoantigen is initially presented by non-B-cell antigen-presenting cells. To test the hypothesis that increased Treg cell activity in B(-/-) mice contributes to their resistance to other autoimmune diseases, WT and B(-/-) NOD mice were given anti-CD25 to transiently deplete CD4(+) CD25(+) Treg cells. The WT and B(-/-) NOD mice given anti-CD25 developed diabetes much earlier than WT mice given rat IgG, whereas rat IgG-treated B(-/-) mice did not develop diabetes. Treg-cell-depleted mice had increased lymphocyte infiltration of the pancreas, salivary glands and thyroid compared with controls given rat IgG. These results are consistent with the hypothesis that resistance of B-cell-deficient NOD mice to several autoimmune diseases is due to the activity of Treg cells.

Culture promotes transfer of thyroid epithelial cell hyperplasia and proliferation by reducing regulatory T cell numbers.

IFN-γ(-/-) NOD.H-2h4 mice develop a spontaneous autoimmune thyroid disease, thyroid epithelial cell hyperplasia and proliferation (TEC H/P) when given NaI in their water for 7+ mo. TEC H/P can be transferred to IFN-γ(-/-) SCID mice by splenocytes from mice with severe (4-5+) disease, and transfer of TEC H/P is improved when splenocytes are cultured prior to transfer. Older (9+ mo) IFN-γ(-/-) NOD.H-2h4 mice have elevated numbers of FoxP3(+) T reg cells, up to 2-fold greater than younger (2 mo) mice. During culture, the number of T reg decreases and this allows the improved transfer of TEC H/P. Co-culture with IL-2 prior to transfer prevents the decrease of T reg and improves their in vitro suppressive ability resulting in reduced TEC H/P in recipient mice. Therefore, culturing splenocytes improves transfer of TEC H/P by reducing the number of T reg and IL-2 inhibits transfer by preserving T reg number and function.

TGF-ß promotes proliferation of thyroid epithelial cells in IFN-γ(-/-) mice by down-regulation of p21 and p27 via AKT pathway.

IFN-γ(-/-) NOD.H-2h4 mice develop an autoimmune disease characterized by hyperplasia and proliferation of thyroid epithelial cells (TEC H/P). Proliferating TECs produce TGF-ß, and IFN-γ inhibits TEC H/P. In the present study, cultured TECs were used to directly determine the mechanisms by which these cytokines act on TECs to result in proliferation or inhibition of proliferation. With TECs from IFN-γ(-/-) NOD.H-2h4 mice or mice expressing the dominant negative TGF-ß type II receptor on TECs, TGF-ß was shown to promote TEC proliferation and IFN-γ was shown to inhibit TEC proliferation in vitro. TGF-ß may promote TEC proliferation by down-regulating antiproliferative molecules p21 and p27, whereas IFN-γ may inhibit proliferation by up-regulating antiproliferative molecules p18 and p21 and down-regulating the pro-proliferative molecule cyclin D. Inhibition of AKT abolished the effect of TGF-ß on p21 and p27, resulting in similar proliferation of TGF-ß-treated and control TECs. Increased expression of proliferating cell nuclear antigen (PCNA), TGF-ß, and p-AKT and decreased expression of p21 and p27 by proliferating TECs correlated with the proliferative state of TEC H/P. Taken together, the results suggest that TGF-ß promotes TEC proliferation by down-regulating p21 and p27 via the AKT pathway in IFN-γ(-/-) NOD.H-2h4 mice, which may have significant implications for development of effective therapeutic strategies targeting the TGF-ß and AKT pathways for treatment of hyperplasia and/or neoplasia.

Transient depletion of B cells in young mice results in activation of regulatory T cells that inhibit development of autoimmune disease in adults.

B-cell depletion therapy can be effective for treating B-cell lymphomas as well as many human and murine autoimmune diseases. B-cell-deficient mice are normally resistant to spontaneous autoimmune thyroiditis (SAT), but they develop SAT if regulatory T cells are transiently depleted during the first 3-6 weeks after birth. This was also a critical time when B-cell depletion effectively inhibited development of SAT in adult mice. The current study was undertaken to test the hypothesis that transient depletion of B cells using anti-CD20 would be sufficient to suppress SAT if B cells were depleted early in life and that inhibition of SAT would be due to the activity of Treg that functioned most effectively when B cells were absent or low. The results presented here support this hypothesis and indicate that development of autoimmune disease in adults is effectively inhibited when anti-CD20 is administered 1-3 weeks after birth. After 3 weeks, transient B-cell depletion is no longer effective, and B-cell depletion must be maintained to effectively suppress autoimmune disease. B-cell depletion in 1- to 3-week-old mice depletes all B-cell subsets, whereas B-cell depletion initiated in adults spares many marginal zone B cells. Following early B-cell depletion, splenic Treg increase in number, and depletion of Treg reverses the inhibitory effect of anti-CD20 on disease development. Early transient depletion of B cells could be useful for preventing autoimmune disease in individuals at high risk for developing autoimmune diseases as adults.

CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis.

CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.

B cell-deficient NOD.H-2h4 mice have CD4+CD25+ T regulatory cells that inhibit the development of spontaneous autoimmune thyroiditis.

Wild-type (WT) NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) when given 0.05% NaI in their drinking water, whereas B cell-deficient NOD.H-2h4 mice are SAT resistant. To test the hypothesis that resistance of B cell-deficient mice to SAT was due to the activity of regulatory CD4+CD25+ T (T reg) cells activated if autoantigen was initially presented on non-B cells, CD25+ T reg cells were transiently depleted in vivo using anti-CD25. B cell-deficient NOD.H-2h4 mice given three weekly injections of anti-CD25 developed SAT 8 wk after NaI water. Thyroid lesions were similar to those in WT mice except there were no B cells in thyroid infiltrates. WT and B cell-deficient mice had similar numbers of CD4+CD25+Foxp3+ cells. Mice with transgenic nitrophenyl-specific B cells unable to secrete immunoglobulin were also resistant to SAT, and transient depletion of T reg cells resulted in severe SAT with both T and B cells in thyroid infiltrates. T reg cells that inhibit SAT were eliminated by day 3 thymectomy, indicating they belong to the subset of naturally occurring T reg cells. However, T reg cell depletion did not increase SAT severity in WT mice, suggesting that T reg cells may be nonfunctional when effector T cells are activated; i.e., by autoantigen-presenting B cells.

Effect of transgenic overexpression of FLIP on lymphocytes on development and resolution of experimental autoimmune thyroiditis.

In our previous studies, resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) was promoted when thyroid epithelial cells were protected from Fas-mediated apoptosis due to transgenic overexpression of FLIP. We hypothesized that if FLIP were overexpressed on lymphocytes, CD4(+) effector cells would be protected from Fas-mediated apoptosis, and resolution would be delayed. To test this hypothesis, we generated transgenic (Tg) mice overexpressing FLIP under the CD2 promoter. Transgenic FLIP was expressed on CD4(+) and CD8(+) T cells and B cells. Transgenic overexpression of FLIP protected cultured splenocytes from Fas-mediated, but not irradiation-induced, apoptosis in vitro. Unexpectedly, Tg(+) donor cells transferred minimal G-EAT, which was partially overcome by depleting donor CD8(+) T cells. When Tg(+) and Tg(-) donors transferred equivalent disease, G-EAT resolution was delayed in FLIP transgenic mice. However, CD2-FLIP Tg(+) donors often transferred less severe G-EAT, even after depletion of CD8(+) T cells. This influenced the rate of G-EAT resolution, resulting in little difference in G-EAT resolution between groups. Tg(+) mice always had reduced anti-mouse thyroglobulin autoantibody responses, compared with Tg(-) littermates, presumably because of FLIP overexpression on B cells. These results suggest that effects of transgenic FLIP on a particular autoimmune disease vary, depending on what cells express the transgene and whether those cells are effector cells or if they function to modulate disease.

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

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

Eosinophils infiltrate thyroids, but have no apparent role in induction or resolution of experimental autoimmune thyroiditis in interferon-gamma(-/-) mice.

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin (MTg)-sensitized splenocytes activated with MTg and interleukin (IL)-12. Our previous studies showed that, when used as donors and recipients, interferon (IFN)-gamma(-/-) and wild-type (WT) DBA/1 mice both develop severe G-EAT. Thyroid lesions in IFN-gamma(-/-) mice have many eosinophils and few neutrophils, while those in WT mice have extensive neutrophil infiltration and few eosinophils. Thyroid lesions in IFN-gamma(-/-) mice consistently resolve by day 40-50, whereas those in WT mice have ongoing inflammation and fibrosis persisting for more than 60 days. To determine if the extensive infiltration of eosinophils in thyroids of IFN-gamma(-/-) mice contributes to thyroid damage and/or early resolution of G-EAT, anti-IL-5 was used to inhibit migration of eosinophils to thyroids. G-EAT severity was compared at day 20 and day 40-50 in IFN-gamma(-/-) recipients given anti-IL-5 or control immunoglobulin G (IgG). Thyroids of anti-IL-5-treated IFN-gamma(-/-) mice had few eosinophils and more neutrophils at day 20, but G-EAT severity scores were comparable to those of control IgG-treated mice at both day 20 and day 40-50. Expression of chemokine (C-X-C motif) ligand 1 (CXCL1) mRNA was higher and that of chemokine (C-C motif) ligand 11 (CCL11) mRNA was lower in thyroids of anti-IL-5-treated IFN-gamma(-/-) mice. IL-5 neutralization did not influence mRNA expression of most cytokines in IFN-gamma(-/-) mice. Thus, inhibiting eosinophil migration to thyroids did not affect G-EAT severity or resolution in IFN-gamma(-/-) mice, suggesting that eosinophil infiltration of thyroids occurs as a consequence of IFN-gamma deficiency, but these cells have no apparent pathogenic role in G-EAT.

TGF-beta promotes thyroid epithelial cell hyperplasia and fibrosis in IFN-gamma-deficient NOD.H-2h4 mice.

IFN-gamma(-/-)NOD.H-2h4 mice given 0.05% NaI in their water develop severe thyroid epithelial cell (thyrocyte) hyperplasia and proliferation (TEC H/P) and fibrosis. Proliferating thyrocytes of IFN-gamma(-/-) mice with TEC H/P produce TGF-beta as demonstrated by immunohistochemical staining and in situ hybridization. Strong expression of activating phosphorylated Smad-2/3 and weak expression of inhibitory Smad-7 by proliferating thyrocytes correlate with the severity of TEC H/P. Splenocytes from IFN-gamma(-/-) mice with severe TEC H/P transfer severe TEC H/P to IFN-gamma(-/-)NOD.H-2h4.SCID mice. Mice given anti-TGF-beta had markedly reduced thyrocyte proliferation and decreased fibrosis compared with mouse Ig-treated controls, suggesting that TGF-beta plays an important role in development of TEC H/P induced by activated splenocytes. Moreover, transgenic IFN-gamma(-/-)NOD.H-2h4 mice expressing TGF-beta on thyrocytes all develop fibrosis and moderate to severe TEC H/P with accelerated kinetics, directly demonstrating a role for TGF-beta in severe TEC H/P and fibrosis.

Interleukin-10 promotes resolution of granulomatous experimental autoimmune thyroiditis.

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin-sensitized splenocytes activated in vitro with mouse thyroglobulin and interleukin (IL)-12. Thyroid lesions reach maximal severity 20 days after cell transfer, and inflammation either resolves or progresses to fibrosis by day 60 depending on the extent of thyroid damage at day 20. Depletion of CD8(+) T cells inhibits G-EAT resolution. Our previous studies indicated that IL-10 was generally higher in G-EAT thyroids that resolved. Using both wild-type and IL-10(-/-) CBA/J mice, this study was undertaken to determine whether G-EAT resolution would be inhibited in the absence of IL-10. The results showed that either depletion of CD8(+) T cells or IL-10 deficiency increased fibrosis and inhibited resolution of inflammation. We also found a correlation between higher expression levels of proinflammatory cytokines and preferential expression levels of proapoptotic molecules, such as FasL and TRAIL, and antiapoptotic molecules, such as FLIP and Bcl-xL, in inflammatory cells from thyroids of both CD8-depleted and IL-10-deficient mice. Furthermore, many of the CD8(+) T cells were also IL-10(+). These results suggest that IL-10 plays an important role in G-EAT resolution and might promote resolution, at least in part, through its production in CD8(+) T cells. Further understanding of the mechanisms that promote the resolution of inflammation will facilitate the development of novel strategies for treating autoimmune diseases.

Cultured murine thyroid epithelial cells expressing transgenic Fas-associated death domain-like interleukin-1beta converting enzyme inhibitory protein are protected from fas-mediated apoptosis.

The antiapoptotic molecule Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP) inhibits Fas-mediated apoptosis by blocking activation of caspase-8. We previously showed that expression of transgenic FLIP on thyroid epithelial cells (TECs) of DBA/1 and CBA/J mice promoted earlier resolution of granulomatous experimental autoimmune thyroiditis in vivo. This study was undertaken to directly determine whether transgenic FLIP expressed on cultured TECs can protect TECs from Fas-mediated apoptosis in vitro. The results indicate that cultured TECs from DBA/1 and CBA/J mice can be sensitized in vitro by interferon-gamma and TNF-alpha to undergo Fas-mediated apoptosis. Transgenic overexpression of FLIP protected cultured TECs of FLIP transgene (Tg)+ DBA/1 and CBA/J mice from Fas-mediated apoptosis, and FLIP small interfering RNA transfection of cultured TECs of FLIP Tg+ DBA/1 and CBA/J mice abolished the protective effect. These in vitro results are consistent with our previous in vivo studies using DBA/1 and CBA/J FLIP Tg+ mice and provide direct support for the hypothesis that transgenic expression of FLIP promotes resolution of granulomatous experimental autoimmune thyroiditis by protecting TECs from apoptosis.

Decreasing TNF-alpha results in less fibrosis and earlier resolution of granulomatous experimental autoimmune thyroiditis.

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced in DBA/1 mice by adoptive transfer of mouse thyroglobulin (MTg)-primed spleen cells. TNF-alpha is an important proinflammatory cytokine and apoptotic molecule involved in many autoimmune diseases. To study its role in G-EAT, anti-TNF-alpha mAb was given to recipient mice. Disease severity was comparable between mice with or without anti-TNF-alpha treatment at days 19-21, the time of maximal severity of G-EAT, suggesting TNF-alpha is not essential for development of thyroid inflammation. However, thyroid lesions resolved at day 48 in anti-TNF-alpha-treated mice, while thyroids of rat Ig-treated controls had fibrosis. These results suggested that reducing TNF-alpha contributed to resolution of inflammation and inhibited fibrosis. Gene and protein expression of inflammatory molecules was examined by RT-PCR and immunostaining, and apoptosis was detected using TUNEL staining and an apoptosis kit. Thyroids of anti-TNF-alpha-treated controls had reduced proinflammatory and profibrotic molecules, e.g., IFN-gamma, IL-1beta, IL-17, inducible NOS and MCP-1, at day 19 compared with thyroids of rat Ig-treated mice. There were more apoptotic thyrocytes in rat Ig-treated controls than in anti-TNF-alpha-treated mice. The site of expression of the anti-apoptotic molecule FLIP also differed between rat Ig-treated and anti-TNF-alpha-treated mice. FLIP was predominantly expressed by inflammatory cells of rat Ig-treated mice and by thyrocytes of anti-TNF-alpha-treated mice. These results suggest that anti-TNF-alpha may regulate expression of proinflammatory cytokines and apoptosis in thyroids, resulting in less inflammation, earlier resolution, and reduced fibrosis.