Granulomatous Experimental Autoimmune Thyroiditis Resolution Research Articles

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.

Eosinophils infiltrate thyroids, but have no apparent role in induction or resolution of experimental autoimmune thyroiditis in interferon‐γ−/− 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.

A critical role for TRAIL in resolution of granulomatous experimental autoimmune thyroiditis

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin (MTG)-sensitized splenocytes activated in vitro with MTG and IL-12. Thyroid lesions reach maximal severity 20 days after cell transfer, and usually resolve or progress to fibrosis by day 60 depending on the extent of thyroid damage at day 20. Our previous studies indicated that neutralization of TNF-alpha or FasL had no effect on G-EAT induction, but neutralization of TNF-alpha promoted, while neutralization of FasL inhibited, G-EAT resolution. TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. This study was undertaken to define the role of endogenous TRAIL in G-EAT development and/or resolution. Neutralization of endogenous TRAIL had little effect on G-EAT induction, but significantly inhibited G-EAT resolution and increased thyroid fibrosis. This correlated with higher expression of pro-inflammatory cytokines and preferential expression of the pro-apoptotic molecule TRAIL, and anti-apoptotic molecules FLIP and Bcl-xL on inflammatory cells in thyroids of anti-TRAIL-treated recipients. The results suggest that endogenous TRAIL is not required for G-EAT development in recipients, but is critical for G-EAT resolution. Endogenous TRAIL might promote resolution, at least in part, through modulation of the balance between pro- and anti-inflammatory cytokines, and the expression pattern of pro- and anti-apoptotic molecules of thyroid epithelial cells (TECs) and inflammatory cells.

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.

Expression of Transgenic FLIP on Thyroid Epithelial Cells Inhibits Induction and Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis in CBA/J Mice

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by transfer of thyroglobulin-primed in vitro activated splenocytes. Thyroid lesions reach maximal severity 20 d later, and inflammation resolves or progresses to fibrosis by d 60, depending on the extent of thyroid damage at d 20. Depletion of CD8+ T cells inhibits G-EAT resolution. We showed that expression of Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP) transgene (Tg) on thyroid epithelial cells (TECs) of DBA/1 mice had no effect on G-EAT induction but promoted earlier resolution of G-EAT. However, when CBA/J wild-type donor cells were transferred to transgenic CBA/J mice expressing FLIP on TECs, they developed less severe G-EAT than FLIP Tg- littermates. Both strains expressed similar levels of the FLIP Tg, but endogenous FLIP was up-regulated to a greater extent on infiltrating T cells during G-EAT development in DBA/1 compared with CBA/J mice. After transient depletion of CD8+ T cells, FLIP Tg+ and Tg- CBA/J recipients both developed severe G-EAT at d 20. Thyroid lesions in CD8-depleted Tg+ recipients were resolving by d 60, whereas lesions in Tg- littermates did not resolve, and most were fibrotic. FLIP Tg+ recipients had increased apoptosis of CD3+ T cells compared with Tg- recipients. The results indicate that transgenic FLIP expressed on TECs in CBA/J mice promotes G-EAT resolution, but induction of G-EAT is inhibited unless CD8+ T cells are transiently depleted.

Expression of transgenic FLIP on thyroid epithelial cells in CBA/J mice inhibits induction and promotes resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) (129.17)

Abstract G-EAT is induced by transfer of thyroglobulin-primed in vitro activated splenocytes. In EAT susceptible DBA/1 and CBA/J mice, inflammation either resolves or progresses to fibrosis by day 60 depending on the extent of damage at day 20. Depletion of CD8+ T cells inhibits G-EAT resolution. We previously showed that FLIP transgene (Tg) expressed on thyroid epithelial cells (TEC) of DBA/1 mice had no effect on induction, but promoted earlier resolution of G-EAT. However, when Tg+ CBA/J mice expressing FLIP on TEC were used as recipients of WT donor splenocytes, they developed less severe G-EAT than FLIP Tg− littermates (average severity score of 1.3 vs. 4.5 at day 20), although both strains had similar levels of FLIP transgene. Following transient depletion of CD8+ T cells, FLIP Tg+ and Tg− CBA/J recipients both developed severe G-EAT (severity score of 4.4) at day 20. Lesions in CD8-depleted Tg+ recipients were resolving by day 60, whereas lesions in Tg− littermates did not resolve and most were fibrotic. FLIP and FasL were mainly expressed by TEC in FLIP Tg+ recipients and by inflammatory cells in Tg− recipients. mRNA expression of IL-10 and IL-13 was higher, and IFN-γ and TNF-α were lower, in FLIP Tg+ compared to Tg− recipients. The results support the hypothesis that transgenic FLIP expressed on TEC in CBA/J mice promotes G-EAT resolution, but induction of G-EAT is inhibited unless CD8+ T cells are depleted (NIH Grant DK35527).

Murine FLIP Transgene Expressed on Thyroid Epithelial Cells Promotes Resolution of Granulomatous Experimental Autoimmune Thyroiditis in DBA/1 Mice

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin-sensitized splenocytes activated in vitro with mouse thyroglobulin and interleukin-12. In wild-type (WT) DBA/1 recipients of WT donor splenocytes, thyroid lesions reach maximal severity at day 20, with ongoing inflammation and extensive fibrosis at day 60. Our previous studies indicated the site of expression of FLIP and Fas ligand [thyroid epithelial cells (TECs) versus inflammatory cells] differed in mice when lesions would resolve or progress to fibrosis. To test the hypothesis that expression of FLIP by TECs would promote earlier G-EAT resolution in DBA/1 mice, transgenic (Tg) DBA/1 mice expressing FLIP on TECs were generated. In FLIP Tg(+) and Tg(-) littermate recipients of WT donor splenocytes, G-EAT severity was comparable at day 20, but fibrosis was decreased, and many lesions resolved by day 60 in Tg(+) but not Tg(-) recipients. FLIP and Fas ligand were primarily expressed by TECs in Tg(+) recipients and by inflammatory cells in Tg(-) recipients at day 60. Apoptosis of inflammatory cells was greater, and expression of proinflammatory cytokines was decreased in thyroids of Tg(+) compared with Tg(-) recipients. These results are consistent with the hypothesis that transgenic expression of FLIP on thyroid epithelial cells promotes earlier resolution of granulomatous experimental autoimmune thyroiditis.

Fas Ligand Is Required for Resolution of Granulomatous Experimental Autoimmune Thyroiditis

We previously suggested that CD8(+) T cells promoted resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) at least in part through regulation of Fas ligand (FasL) expression on thyroid epithelial cells. To directly evaluate the role of the Fas pathway in G-EAT resolution, Fas- and FasL-deficient mice on the NOD.H-2h4 background were used as recipients of activated G-EAT effector cells. When MTg-primed wild-type (WT) donor splenocytes were activated and transferred to WT recipients, thyroid lesions reached maximal severity on day 20 and resolved on day 50. Fas, FasL, and FLIP were up-regulated, and many apoptotic inflammatory cells were detected in recipient thyroids on day 20. Fas was predominantly expressed by inflammatory cells, and FasL and FLIP were mainly expressed by thyroid epithelial cells. After depletion of CD8(+) T cells, G-EAT resolution was delayed, FLIP and FasL were predominantly expressed by inflammatory cells, and few inflammatory cells were apoptotic. When WT donor splenocytes were transferred to gld recipients, disease severity on day 20 was similar to that in WT recipients, but resolution was delayed. As in CD8-depleted WT recipients, there were few apoptotic inflammatory cells, and FLIP and FasL were expressed primarily by inflammatory cells. These results indicated that the expression of functional FasL in recipient mice was critical for G-EAT resolution. WT cells induced minimal disease in lpr recipients. This was presumably because donor cells were eliminated by the increased FasL on lpr recipient cells, because donor cells were not eliminated, and the mice developed G-EAT if lpr recipients were given anti-FasL mAb.

FLIP and FasL expression by inflammatory cells vs thyrocytes can be predictive of chronic inflammation or resolution of autoimmune thyroiditis

Spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice is a model of chronic inflammation of the thyroid, while granulomatous experimental autoimmune thyroiditis (G-EAT) is a model with spontaneous resolution of inflammation. In chronic inflammation (SAT), Fas, FasL, and FLIP were upregulated and predominant in inflammatory cells. There were few apoptotic cells, and low expression of active caspase-8 and -3. In resolving G-EAT in CBA/J and NOD.H-2h4 mice, FasL and FLIP were predominantly expressed by thyrocytes. There were many apoptotic inflammatory cells, and increased expression of active caspase-8 and -3. Depletion of CD8+ T cells inhibited G-EAT resolution and resulted in chronic inflammation. FLIP was expressed predominantly by inflammatory cells, and apoptosis of inflammatory cells and expression of active caspase-3 was reduced as in chronic SAT. Thus, differences in expression of pro- or antiapoptotic molecules in SAT or G-EAT were apparently related to the acute vs chronic nature of the inflammatory response rather than the method of disease induction. Upregulation of FLIP by inflammatory cells may block Fas-mediated apoptosis, contributing to chronic inflammation, whereas increased FLIP expression by thyrocytes in resolving G-EAT may protect thyrocytes from apoptosis, and FasL expression by thyrocytes may induce apoptosis of inflammatory cells, contributing to resolution.