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A defect in tryptophan catabolism impairs tolerance in nonobese diabetic mice.

Grohmann U, Fallarino F, Bianchi R, Orabona C, Vacca C, Fioretti MC, Puccetti P - J. Exp. Med. (2003)

Bottom Line: This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism.This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production.This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine, University of Perugia, 06126 Perugia, Italy. ugrohmann@tin.it

ABSTRACT
The predisposition of nonobese diabetic (NOD) mice to develop autoimmunity reflects deficiencies in both peripheral and central tolerance. Several defects have been described in these mice, among which aberrant antigen-presenting cell function and peroxynitrite formation. Prediabetes and diabetes in NOD mice have been targeted with different outcomes by a variety of immunotherapies, including interferon (IFN)-gamma. This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism. Here, we provide evidence that IFN-gamma fails to induce tolerizing properties in dendritic cells from highly susceptible female mice early in prediabetes. This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production. However, the use of a peroxynitrite inhibitor can rescue tryptophan catabolism and tolerance in those mice. This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.

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Failure of IFN-γ to induce IDO expression and activity in early prediabetic NOD female mice. (A) Functional IDO activity in response to IFN-γ was measured in vitro in terms of the ability to metabolize tryptophan to kynurenine with the use of DCs recovered from NOD female and male mice of two different ages (4 or 8 wk) or control C57BL/6 female mice. Kynurenine levels in supernatants were measured by HPLC, and results are the mean ± SD of triplicate samples in one of three experiments. (B) IDO expression in DCs was assessed by Western blot. DCs were recovered from NOD female or male mice or control DBA/2 females and treated overnight with IFN-γ, and IDO expression was investigated with an IDO-specific antibody. The positive control consisted of IDO-expressing MC24 transfectants and the negative control consisted of mock-transfected MC22 cells. Loading controls (not shown in the figure) consisted of samples reprobed with β-actin-specific antibody. One experiments of three. (C) Stat1 phosphorylation in response to IFN-γ was studied in NOD female and male mice at 4 wk and in age-matched control C57BL/6 female mice. DC lysates were resolved on SDS-PAGE and immunoblotted with anti-phosphoStat1 and then reblotted with anti-Stat1, reblots showing equal loading of Stat1 in each set of lanes. One of three experiments with similar results.
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fig2: Failure of IFN-γ to induce IDO expression and activity in early prediabetic NOD female mice. (A) Functional IDO activity in response to IFN-γ was measured in vitro in terms of the ability to metabolize tryptophan to kynurenine with the use of DCs recovered from NOD female and male mice of two different ages (4 or 8 wk) or control C57BL/6 female mice. Kynurenine levels in supernatants were measured by HPLC, and results are the mean ± SD of triplicate samples in one of three experiments. (B) IDO expression in DCs was assessed by Western blot. DCs were recovered from NOD female or male mice or control DBA/2 females and treated overnight with IFN-γ, and IDO expression was investigated with an IDO-specific antibody. The positive control consisted of IDO-expressing MC24 transfectants and the negative control consisted of mock-transfected MC22 cells. Loading controls (not shown in the figure) consisted of samples reprobed with β-actin-specific antibody. One experiments of three. (C) Stat1 phosphorylation in response to IFN-γ was studied in NOD female and male mice at 4 wk and in age-matched control C57BL/6 female mice. DC lysates were resolved on SDS-PAGE and immunoblotted with anti-phosphoStat1 and then reblotted with anti-Stat1, reblots showing equal loading of Stat1 in each set of lanes. One of three experiments with similar results.

Mentions: The tolerogenic properties of CD8+ DCs are dependent on the activation of immunosuppressive tryptophan catabolism, which may affect the proliferation and survival of NRP-A7-specific T cells (13, 17). To gain insight into the mechanism underlying IFN-γ unresponsiveness in CD8+ DCs from 4-wk-old female mice, we measured IDO functional activity in terms of ability to metabolize tryptophan to kynurenine in vitro (Fig. 2 A). On comparing female and male mice of 4 or 8 wk of age, we found a selective inability of IFN-γ to induce tryptophan catabolism in female mice at 4 wk. Both NOD male mice and control C57BL/6 female mice were susceptible to the enhancing effect of IFN-γ at 4 and 8 wk. To investigate whether the defective IDO expression of female mice at 4 wk was due to inhibition of enzyme functional activity or to impaired gene transcription, we analyzed IDO expression by Western blot using rabbit polyclonal IDO-specific antibody (Fig. 2 B). Considerable expression of the enzyme protein was induced by activation with IFN-γ in CD8+ DCs from 4- or 8-wk-old NOD male mice as well as DBA/2 controls. In NOD females, IDO expression was induced by IFN-γ at 8 but not 4 wk of age. Thus, it appeared that IFN-γ would not cause IDO expression in 4-wk-old female mice. This could be due to a specific defect in the IFN-γ signaling mechanism regulating IDO expression in CD8+ DCs. Because Stat1 phosphorylation is required for IFN-γ-induced transcription of the IDO gene (10), we measured Stat1 phosphorylation in female versus male mice at 4 wk (Fig. 2 C). In contrast to NOD male mice and C57BL/6 controls, no Stat1 phosphorylation was observed in NOD females in response to IFN-γ. Thus, the failure of IFN-γ to induce IDO expression in CD8+ DCs from early prediabetic female mice appeared to be associated with defective phosphorylation of Stat1.


A defect in tryptophan catabolism impairs tolerance in nonobese diabetic mice.

Grohmann U, Fallarino F, Bianchi R, Orabona C, Vacca C, Fioretti MC, Puccetti P - J. Exp. Med. (2003)

Failure of IFN-γ to induce IDO expression and activity in early prediabetic NOD female mice. (A) Functional IDO activity in response to IFN-γ was measured in vitro in terms of the ability to metabolize tryptophan to kynurenine with the use of DCs recovered from NOD female and male mice of two different ages (4 or 8 wk) or control C57BL/6 female mice. Kynurenine levels in supernatants were measured by HPLC, and results are the mean ± SD of triplicate samples in one of three experiments. (B) IDO expression in DCs was assessed by Western blot. DCs were recovered from NOD female or male mice or control DBA/2 females and treated overnight with IFN-γ, and IDO expression was investigated with an IDO-specific antibody. The positive control consisted of IDO-expressing MC24 transfectants and the negative control consisted of mock-transfected MC22 cells. Loading controls (not shown in the figure) consisted of samples reprobed with β-actin-specific antibody. One experiments of three. (C) Stat1 phosphorylation in response to IFN-γ was studied in NOD female and male mice at 4 wk and in age-matched control C57BL/6 female mice. DC lysates were resolved on SDS-PAGE and immunoblotted with anti-phosphoStat1 and then reblotted with anti-Stat1, reblots showing equal loading of Stat1 in each set of lanes. One of three experiments with similar results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196078&req=5

fig2: Failure of IFN-γ to induce IDO expression and activity in early prediabetic NOD female mice. (A) Functional IDO activity in response to IFN-γ was measured in vitro in terms of the ability to metabolize tryptophan to kynurenine with the use of DCs recovered from NOD female and male mice of two different ages (4 or 8 wk) or control C57BL/6 female mice. Kynurenine levels in supernatants were measured by HPLC, and results are the mean ± SD of triplicate samples in one of three experiments. (B) IDO expression in DCs was assessed by Western blot. DCs were recovered from NOD female or male mice or control DBA/2 females and treated overnight with IFN-γ, and IDO expression was investigated with an IDO-specific antibody. The positive control consisted of IDO-expressing MC24 transfectants and the negative control consisted of mock-transfected MC22 cells. Loading controls (not shown in the figure) consisted of samples reprobed with β-actin-specific antibody. One experiments of three. (C) Stat1 phosphorylation in response to IFN-γ was studied in NOD female and male mice at 4 wk and in age-matched control C57BL/6 female mice. DC lysates were resolved on SDS-PAGE and immunoblotted with anti-phosphoStat1 and then reblotted with anti-Stat1, reblots showing equal loading of Stat1 in each set of lanes. One of three experiments with similar results.
Mentions: The tolerogenic properties of CD8+ DCs are dependent on the activation of immunosuppressive tryptophan catabolism, which may affect the proliferation and survival of NRP-A7-specific T cells (13, 17). To gain insight into the mechanism underlying IFN-γ unresponsiveness in CD8+ DCs from 4-wk-old female mice, we measured IDO functional activity in terms of ability to metabolize tryptophan to kynurenine in vitro (Fig. 2 A). On comparing female and male mice of 4 or 8 wk of age, we found a selective inability of IFN-γ to induce tryptophan catabolism in female mice at 4 wk. Both NOD male mice and control C57BL/6 female mice were susceptible to the enhancing effect of IFN-γ at 4 and 8 wk. To investigate whether the defective IDO expression of female mice at 4 wk was due to inhibition of enzyme functional activity or to impaired gene transcription, we analyzed IDO expression by Western blot using rabbit polyclonal IDO-specific antibody (Fig. 2 B). Considerable expression of the enzyme protein was induced by activation with IFN-γ in CD8+ DCs from 4- or 8-wk-old NOD male mice as well as DBA/2 controls. In NOD females, IDO expression was induced by IFN-γ at 8 but not 4 wk of age. Thus, it appeared that IFN-γ would not cause IDO expression in 4-wk-old female mice. This could be due to a specific defect in the IFN-γ signaling mechanism regulating IDO expression in CD8+ DCs. Because Stat1 phosphorylation is required for IFN-γ-induced transcription of the IDO gene (10), we measured Stat1 phosphorylation in female versus male mice at 4 wk (Fig. 2 C). In contrast to NOD male mice and C57BL/6 controls, no Stat1 phosphorylation was observed in NOD females in response to IFN-γ. Thus, the failure of IFN-γ to induce IDO expression in CD8+ DCs from early prediabetic female mice appeared to be associated with defective phosphorylation of Stat1.

Bottom Line: This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism.This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production.This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine, University of Perugia, 06126 Perugia, Italy. ugrohmann@tin.it

ABSTRACT
The predisposition of nonobese diabetic (NOD) mice to develop autoimmunity reflects deficiencies in both peripheral and central tolerance. Several defects have been described in these mice, among which aberrant antigen-presenting cell function and peroxynitrite formation. Prediabetes and diabetes in NOD mice have been targeted with different outcomes by a variety of immunotherapies, including interferon (IFN)-gamma. This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism. Here, we provide evidence that IFN-gamma fails to induce tolerizing properties in dendritic cells from highly susceptible female mice early in prediabetes. This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production. However, the use of a peroxynitrite inhibitor can rescue tryptophan catabolism and tolerance in those mice. This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.

Show MeSH
Related in: MedlinePlus