Loss of T-bet, but not STAT1, prevents the development of experimental autoimmune encephalomyelitis - PubMed (original) (raw)

Loss of T-bet, but not STAT1, prevents the development of experimental autoimmune encephalomyelitis

Estelle Bettelli et al. J Exp Med. 2004.

Abstract

The transcription factors signal transducer and activator of transcription (STAT)1 and T-bet control the differentiation of interferon (IFN)-gamma-producing T helper type (Th)1 cells. Here we compare the role of T-bet and STAT1 in the initiation and regulation of experimental autoimmune encephalomyelitis (EAE), a disease initiated by Th1 cells. T-bet-deficient mice immunized with myelin oligodendrocyte glycoprotein (MOG) were resistant to the development of EAE. This protection was also observed when T-bet(-/-) mice were crossed to the MOG-specific 2D2 T cell receptor transgenic strain. In contrast, although T-bet is downstream of STAT1, STAT1(-/-) mice were highly susceptible to EAE and developed more severe and accelerated disease with atypical neuropathologic features. The function of T-bet was dominant as mice deficient in both T-bet and STAT1 were also protected from EAE. CD4(+) CD25(+) regulatory T cells from these two mice strains were fully competent and do not explain the difference in disease susceptibility. However, enhanced EAE in STAT1(-/-) mice was associated with continued generation of IFN-gamma-producing Th1 cells and up-regulation of selective chemokines responsible for the increased recruitment of macrophages and neutrophils in the central nervous system. Although the two transcription factors, STAT1 and T-bet, both induce IFN-gamma gene transcription, our results demonstrate marked differences in their function in regulating pathogenic Th1 cell responses.

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Figures

Figure 1.

Figure 1.

T-bet–deficient mice are resistant to EAE induction, whereas STAT1−/− mice develop fulminant EAE. (A) EAE development in T-bet– and STAT1-deficient mice. Groups of T-bet−/− and T-bet−/+ mice and STAT1−/− and 129 wild-type mice (6–7 mice per group) were immunized with MOG 35-55 and injected with pertussis toxin (a and c). T-bet−/− or T-bet+/+ 2D2 MOG-specific TCR transgenic mice were injected with pertussis toxin (b). Mice were observed daily for the development of EAE. (B) Histology of 2D2 TCR transgenic mice with spontaneous EAE. (a) Posterior spinal cord of a 2D2 × T-bet+/+ mouse with spontaneous EAE. There are small numbers of typical mononuclear cell perivascular infiltrates in leptomeninges and parenchyma (arrows). There were similar infiltrates in 2D2 × STAT1+/+ mice (not depicted). Magnification, 57. (b) Posterior spinal cord of 2D2 × T-bet−/− mouse. There are no inflammatory lesions. Magnification, 57. (c) Posterior spinal cord of 2D2 × STAT1−/− mouse with severe clinical disease. There is a large symmetric destructive inflammatory lesion in the posterior columns. Inset, higher power demonstrates confluent parenchymal inflammatory cell infiltration, predominantly by neutrophils. Magnification, 57; magnification of inset, 450. (d) Adjacent section to (c) demonstrating absence of axons in lesion center. Inset, higher power demonstrates neutrophils and complete loss of axons. Intact black fibers are axons on the periphery of the lesion. Magnification, 57; magnification of inset, 450. (e) Brain stem of 2D2 × STAT1−/− mouse with atypical disease. A large subpial/subependymal inflammatory lesion adjacent to the lateral foramen in the pons fills most of the field. Myelinated fibers (blue) are preserved in the lower left. *, cerebellum; magnification, 71. (f) Higher power of brain stem lesion in (e) with fibrin exudation from a vessel (arrow). Neutrophils and foamy macrophages are the predominant inflammatory cells. Magnification, 286. a–c, e, and f, Luxol fast blue/hematoxylin and eosin stain; d, Bielschowsky silver impregnation for axons.

Figure 1.

Figure 1.

T-bet–deficient mice are resistant to EAE induction, whereas STAT1−/− mice develop fulminant EAE. (A) EAE development in T-bet– and STAT1-deficient mice. Groups of T-bet−/− and T-bet−/+ mice and STAT1−/− and 129 wild-type mice (6–7 mice per group) were immunized with MOG 35-55 and injected with pertussis toxin (a and c). T-bet−/− or T-bet+/+ 2D2 MOG-specific TCR transgenic mice were injected with pertussis toxin (b). Mice were observed daily for the development of EAE. (B) Histology of 2D2 TCR transgenic mice with spontaneous EAE. (a) Posterior spinal cord of a 2D2 × T-bet+/+ mouse with spontaneous EAE. There are small numbers of typical mononuclear cell perivascular infiltrates in leptomeninges and parenchyma (arrows). There were similar infiltrates in 2D2 × STAT1+/+ mice (not depicted). Magnification, 57. (b) Posterior spinal cord of 2D2 × T-bet−/− mouse. There are no inflammatory lesions. Magnification, 57. (c) Posterior spinal cord of 2D2 × STAT1−/− mouse with severe clinical disease. There is a large symmetric destructive inflammatory lesion in the posterior columns. Inset, higher power demonstrates confluent parenchymal inflammatory cell infiltration, predominantly by neutrophils. Magnification, 57; magnification of inset, 450. (d) Adjacent section to (c) demonstrating absence of axons in lesion center. Inset, higher power demonstrates neutrophils and complete loss of axons. Intact black fibers are axons on the periphery of the lesion. Magnification, 57; magnification of inset, 450. (e) Brain stem of 2D2 × STAT1−/− mouse with atypical disease. A large subpial/subependymal inflammatory lesion adjacent to the lateral foramen in the pons fills most of the field. Myelinated fibers (blue) are preserved in the lower left. *, cerebellum; magnification, 71. (f) Higher power of brain stem lesion in (e) with fibrin exudation from a vessel (arrow). Neutrophils and foamy macrophages are the predominant inflammatory cells. Magnification, 286. a–c, e, and f, Luxol fast blue/hematoxylin and eosin stain; d, Bielschowsky silver impregnation for axons.

Figure 2.

Figure 2.

CD4+ CD25+ from both T-bet– and STAT1-deficient mice can suppress the proliferation of effector CD4+ T cells. CD4+ CD25+ and CD4+ CD25− T cells from STAT1−/−, T-bet−/−, and respective wild-type littermates were purified from the spleens and lymph nodes as described in Materials and Methods. Proliferation of CD4+ CD25− cells in the presence of irradiated APCs, 1 μg/ml anti-CD3 antibody, and different ratio of CD4+ CD25+ T cells (from STAT1 knockout, bold hatched bars; STAT1 wild-type, hatched bars; T-bet knockout, closed bars; T-bet wild-type, dotted bars) was determined by thymidine incorporation. These results are representative of four independent experiments.

Figure 3.

Figure 3.

In absence of T-bet, STAT1−/− mice are protected from EAE. Groups of T-bet−/− (n = 14), STAT1−/− (n = 13), and STAT1−/− T-bet−/− (n = 13) mice were immunized with MOG 35-55 and injected with pertussis toxin. The development of EAE was followed over time. The data represent two independent experiments.

Figure 4.

Figure 4.

Proliferative response and cytokine production of T cells from T-bet– and STAT1-deficient mice. CD4+ T cells from naive 2D2 × STAT1−/−, 2D2 × T-bet−/−, 2D2 (T-bet × STAT1)−/− mice, and relative wild-type littermates were stimulated in vitro with MOG 35-55 peptide and syngeneic APCs. Proliferation was measured 72 h later by [3H]thymidine incorporation (A). The data are representative of at least four independent experiments. IFN-γ, IL−10, IL-5, IL-4, and TGF-β (B) produced in the culture supernatant was measured by ELISA 48 h after stimulation with peptide MOG 35-55 and APCs. (C) Ratio of IL-10/IFN-γ production in CD4+ T cells from 2D2 × T-bet−/−, 2D2 × T-bet+/+, 2D2 × STAT1−/−, 2D2 × STAT1+/+, 2D2 (T-bet × STAT1)−/−, and 2D2 (T-bet × STAT1)+/+. The data are presented as the average between three representative experiments ± SE.

Figure 5.

Figure 5.

Chemokine expression in the brain of T-bet– and STAT1-deficient mice during EAE. Groups of STAT1−/− and STAT1+/+ mice (five mice/group) were immunized with MOG 35-55 and pertussis toxin. During the first attack of EAE, the brain from these mice was removed and chemokine expression was determined by real-time PCR. The results represent the mean relative mRNA expression (2-ΔΔCT × 1,000) of a particular chemokine within a group ± SE between samples of this group.

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