Shiguang Yu

Early Requirement for B Cells for Development of Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice

B cells are known to play an important role in the pathogenesis of several autoimmune diseases. NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) and anti-mouse thyroglobulin (MTg) autoantibodies, the levels of which correlate closely with the severity of thyroid lesions. NOD.H-2h4 mice genetically deficient in B cells (NOD.Kμnull) or rendered B cell-deficient by treatment from birth with anti-IgM develop minimal SAT. B cells were required some time in the first 4–6 wk after birth, because NOD.Kμnull or NOD.H-2h4 mice did not develop SAT when they were reconstituted with B cells as adults. The requirement for B cells was apparently not solely to produce anti-MTg autoantibodies, because passive transfer of anti-MTg Ab did not enable B cell-deficient mice to develop SAT, and mice given B cells as adults produced autoantibodies but did not develop SAT. B cell-deficient mice developed SAT if their T cells developed from bone marrow precursors in the presence of B cells. Because B cells are required early in life and their function cannot be replaced by anti-MTg autoantibodies, B cells may be required for the activation or selection of autoreactive T cells. These autoreactive T cells are apparently unable to respond to Ag if B cells are absent in the first 4–6 wk after birth.

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.

Dual Roles for IFN-γ, But Not for IL-4, in Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice

Spontaneous autoimmune thyroiditis (SAT) is an organ-specific autoimmune disease characterized by chronic inflammation of the thyroid by T and B lymphocytes. To investigate the roles of Th1 and Th2 cytokines in the pathogenesis of SAT, IFN-γ−/− and IL-4−/− NOD.H-2h4 mice were generated. IL-4−/− mice developed lymphocytic SAT (L-SAT) comparable to that of wild-type (WT) mice. They produced little anti-mouse thyroglobulin (MTg) IgG1, but had levels of anti-MTg IgG2b comparable to WT mice. Compared with WT mice, IFN-γ−/− mice produced significantly less anti-MTg IgG1 and IgG2b. Absence of IFN-γ resulted in abnormal proliferation of thyroid epithelial cells with minimal lymphocyte infiltration. Thyroids of IFN-γ−/− mice had markedly reduced B lymphocyte chemoattractant expression, B cell and plasma cell infiltration, and decreased MHC class II expression on thyrocytes compared with WT mice. Adoptive transfer of WT splenocytes to IFN-γ−/− mice restored the capacity to develop typical L-SAT, enhanced anti-MTg IgG1 and IgG2b production, up-regulated MHC class II expression on thyrocytes and decreased thyrocyte proliferation. These results suggest that IFN-γ plays a dual role in the development of SAT. IFN-γ is required for development of L-SAT, and it also functions to inhibit thyroid epithelial cell proliferation.

B cell depletion inhibits spontaneous autoimmune thyroiditis in NOD.H-2h4 mice.

B cells are important for the development of most autoimmune diseases. B cell depletion immunotherapy has emerged as an effective treatment for several human autoimmune diseases, although it is unclear whether B cells are necessary for disease induction, autoantibody production, or disease progression. To address the role of B cells in a murine model of spontaneous autoimmune thyroiditis (SAT), B cells were depleted from adult NOD.H-2h4 mice using anti-mouse CD20 mAb. Anti-CD20 depleted most B cells in peripheral blood and cervical lymph nodes and 50–80% of splenic B cells. Flow cytometry analysis showed that marginal zone B cells in the spleen were relatively resistant to depletion by anti-CD20, whereas most follicular and transitional (T2) B cells were depleted after anti-CD20 treatment. When anti-CD20 was administered before development of SAT, development of SAT and anti-mouse thyroglobulin autoantibody responses were reduced. Anti-CD20 also reduced SAT severity and inhibited further increases in anti-mouse thyroglobulin autoantibodies when administered to mice that already had autoantibodies and thyroid inflammation. The results suggest that B cells are necessary for initiation as well as progression or maintenance of SAT in NOD.H-2h4 mice.

Comparison of sensitivity of Th1, Th2, and Th17 cells to Fas-mediated apoptosis

Following activation through the TCR, CD4+ T cells can differentiate into three major subsets: Th1, Th2, and Th17 cells. IL‐17‐secreting Th17 cells play an important role in the pathogenesis of several autoimmune diseases and in immune responses to pathogens, but little is known about the regulation of apoptosis in Th17 cells. In this study, the sensitivity of in vitro‐polarized Th1, Th2, and Th17 cells to Fas‐mediated apoptosis was compared directly by different methods. The order of sensitivity of T cell subsets to Fas‐mediated apoptosis is: Th1 > Th17 > Th2. The greater sensitivity of Th17 cells to Fas‐mediated apoptosis compared with Th2 cells correlated with their higher expression of FasL and comparable expression of the antiapoptotic molecule FLIP. The decreased sensitivity of Th17 compared with Th1 cells correlated with the higher expression of FLIP by Th17 cells. Transgenic overexpression of FLIP in T cells protected all three subsets from Fas‐mediated apoptosis. These findings provide new knowledge for understanding how survival of different subsets of T cells is regulated.

Role of TGFβ in Development of Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice

Nearly 100% of NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) and produce anti-mouse thyroglobulin autoantibodies when they receive 0.05% NaI in their drinking water beginning at 8 wk of age. Our previous studies showed that TGFβ1 mRNA was constitutively expressed in thyroids and spleens of normal NOD.H-2h4 mice but not other strains of mice. To determine whether TGFβ might have a role in SAT, mice were given anti-TGFβ mAb at various times during development of SAT. Anti-TGFβ markedly inhibited development of SAT and production of anti-mouse thyroglobulin IgG1 autoantibodies. Anti-TGFβ was most effective in inhibiting SAT when given during the time thyroid lesions were developing, i.e., starting 4 wk after administration of NaI water. The active form of the TGFβ1 protein was present in thyroids of mice with SAT but not in normal NOD.H-2h4 thyroids. However, thyrocytes of normal NOD.H-2h4 thyroids did express latent TGFβ1. TGFβ1 protein expression in the thyroid correlated with SAT severity scores, and administration of anti-TGFβ inhibited TGFβ1 protein expression in both the thyroid and spleen. TGFβ1 was produced primarily by inflammatory cells and was primarily localized in areas of the thyroid containing clusters of CD4+ T and B cells. Depletion of CD8+ T cells had no effect on TGFβ1 protein expression. Activation of splenic T cells was apparently not inhibited by anti-TGFβ, because up-regulation of mRNA for cytokines and other T cell activation markers was similar for control and anti-TGFβ-treated mice. TGFβ1 may function by promoting migration to, or retention of, inflammatory cells in the thyroid.

Thyrocytes Responding to IFN-γ Are Essential for Development of Lymphocytic Spontaneous Autoimmune Thyroiditis and Inhibition of Thyrocyte Hyperplasia

IFN-γ promotes the development of lymphocytic spontaneous autoimmune thyroiditis (L-SAT) in NOD.H-2h4 mice and inhibits the development of thyrocyte hyperplasia and proliferation (TEC H/P). The precise mechanisms by which IFN-γ promotes L-SAT and inhibits TEC H/P are unknown. To determine whether responsiveness of lymphocytes or thyrocytes to IFN-γ is important for the development of these lesions, IFN-γR−/− mice, which develop TEC H/P similar to IFN-γ−/− mice, were used as recipients for adoptive cell transfer. Wild-type (WT) splenocytes or bone marrow induced L-SAT and inhibited TEC H/P in IFN-γ−/−, but not IFN-γR−/− recipients. IFN-γR−/− recipients of WT cells developed severe TEC H/P, but did not develop L-SAT, suggesting that thyrocytes responding to IFN-γ are important for inhibition of TEC H/P. Unexpectedly, IFN-γR−/− splenocytes or bone marrow did not induce L-SAT in IFN-γ−/− or WT mice even though IFN-γR−/− lymphocyte donors produced as much IFN-γ as lymphocytes from WT donors, and thyrocytes could respond to IFN-γ. Real-time PCR indicated that recipients of IFN-γR−/− bone marrow expressed less mRNA for IFN-γ-inducible chemokines compared with recipients of WT bone marrow. This might limit the migration of IFN-γR−/− lymphocytes to thyroids. Few IFN-γR−/− lymphocytes infiltrated thyroids even in the presence of WT lymphocytes, suggesting that lymphocytes unable to respond to IFN-γ are not induced to migrate to thyroids. These results suggest that thyrocytes must be able to respond to IFN-γ for the development of L-SAT and inhibition of TEC H/P, and lymphocytes must be able to respond to IFN-γ to induce L-SAT.

TGF-β 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-β, 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-β type II receptor on TECs, TGF-β was shown to promote TEC proliferation and IFN-γ was shown to inhibit TEC proliferation in vitro. TGF-β 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-β on p21 and p27, resulting in similar proliferation of TGF-β-treated and control TECs. Increased expression of proliferating cell nuclear antigen (PCNA), TGF-β, 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-β 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-β 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.