Mechanisms of Spontaneous Resolution versus Fibrosis in Granulomatous Experimental Autoimmune Thyroiditis

Abstract

When granulomatous experimental autoimmune thyroiditis (G-EAT) was induced in CBA/J or DBA/1 mice, thyroid lesions resolved in less severe (3+) G-EAT in wild-type mice or severe (5+) G-EAT in IFN-γ−/− mice, but progressed to fibrosis in 5+ G-EAT in wild-type mice. To define the mechanisms leading to these distinct outcomes, the expression of inflammatory and apoptotic molecules and infiltrating cells was evaluated using immunohistochemistry, RT-PCR, and confocal microscopy. The ratio of CD4+/CD8+ T cells in thyroid infiltrates was one factor that predicted G-EAT outcome. CD4+ T cells outnumbered CD8+ T cells when lesions progressed to fibrosis, while CD8+ T cells outnumbered CD4+ T cells in thyroids that resolved. Fas, Fas ligand, FLIP, TNF-α, inducible NO synthase, TGF-β, and IFN-γ were highly expressed by infiltrating cells when G-EAT progressed to fibrosis. The expression of active caspase-3 was low, possibly contributing to the persistence of CD4+ T cells in fibrosis. In contrast, FLIP was mainly expressed by thyrocytes in resolving G-EAT, the expression of active caspase-3 was high, and resolution correlated with apoptosis of infiltrating cells. There was also relatively less expression of TGF-β, IFN-γ, TNF-α, and inducible NO synthase and higher expression of IL-10 in resolving G-EAT than in G-EAT that progressed to fibrosis. These differences were particularly striking when comparing IFN-γ−/− vs wild-type mice. These results suggest that several opposing biological mechanisms contribute to the outcome of an ongoing autoimmune response. These include differential expression of pro- and antiapoptotic molecules, cytokines, and the ratio of CD4+ vs CD8+ T cells.

Effect of Transgenic Overexpression of FLIP on Lymphocytes on Development and Resolution of Experimental Autoimmune Thyroiditis

“…Clearly, CD2-FLIP transgenic mice produced less anti-MTg autoantibody than their Tg Ϫ littermates (Table 1), and susceptibility to EAT and G-EAT is known to correlate with levels of anti-MTg autoantibody responses. [3][4][5][6][7][8] However, it is not clear why protection of B cells from Fas-mediated apoptosis due to overexpression of FLIP would lead to reduced autoantibody responses. Further studies will be required to determine how overexpression of FLIP in B cells might contribute to the greater resistance to G-EAT in CD2-FLIP transgenic mice.…”

“…7,21,23,[32][33][34] Proinflammatory cytokines such as IFN-␥ and TNF-␣ promote inflammation, [32][33][34] and can sensitize thyroid epithelial cells to undergo Fas-mediated apoptosis, 9,[32][33][34]36 whereas anti-inflammatory cytokines such as IL-10 inhibit inflammation 7,21,23,29 and promote apoptosis of inflammatory cells. 9 CD2-FLIP transgenic mice produced lower levels of both proinflammatory IFN-␥ and IL-17 cytokines, as well as the antiinflammatory cytokine IL-10, compared with their Tg Ϫ littermates (Table 2 and Figures 5 and 6).…”

“…[3][4][5][6][7] Serum dilution is reported in Table 1. Normal mouse serum used at the same dilutions always gave an optical density value of OD Ͻ 0.05.…”

“…7,23,29,[32][33][34] Others have shown that immune responses in CD2-FLIP transgenic mice are biased toward Th2 responses 27 although this was overcome to some extent by activating cells under Th1 polarization conditions. 35 To determine whether pro-and anti-inflammatory cytokines were differentially expressed in splenocytes from CD2-FLIP Tg ϩ and Tg Ϫ donors after in vitro activation, mRNA expression of cytokines in splenocytes from MTg-immunized Tg ϩ and Tg Ϫ mice activated with MTg and IL-12 (Th1 polarization conditions) was determined by RT-PCR ( Figure 5).…”

“…[3][4][5][6][7] Thyroid lesions reach maximal severity 20 days after cell transfer, and inflammation either resolves or progresses to fibrosis at day 50 to day 60, depending on the extent of damage at day 20. [3][4][5][6][7] DBA/1 recipients typically develop very severe thyroid lesions (5ϩ severity score) by day 20, with few or no remaining intact follicles, and inflammation and fibrosis persist 60 days after cell transfer. 4 -7 CBA/J recipients also develop very severe G-EAT, but there are usually some intact thyroid follicles, less neutrophil infiltration, and less fibrosis at day 20, compared with lesions in DBA/1 mice.…”