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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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Ability of GED to restore IFN-γ responsiveness in CD8+ DCs from early prediabetic NOD female mice. (A) GED was examined for ability to restore IDO expression and Stat1 phosphorylation in response to IFN-γ. CD8+ DCs from 4-wk-old NOD female mice were treated overnight with IFN-γ and assayed for IDO expression or Stat1 phosphorylation. One experiment representative of three. (B) GED was also studied for ability to restore kynurenine production by CD8+ DCs from the same donors. One experiment representative of three.
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fig4: Ability of GED to restore IFN-γ responsiveness in CD8+ DCs from early prediabetic NOD female mice. (A) GED was examined for ability to restore IDO expression and Stat1 phosphorylation in response to IFN-γ. CD8+ DCs from 4-wk-old NOD female mice were treated overnight with IFN-γ and assayed for IDO expression or Stat1 phosphorylation. One experiment representative of three. (B) GED was also studied for ability to restore kynurenine production by CD8+ DCs from the same donors. One experiment representative of three.

Mentions: In NOD mice, activated macrophages produce high amounts of nitric oxide (NO), which is thought to contribute to the destructive phase of insulitis (19, 20). There is evidence that tyrosine nitration of Stat1 by endogenous NO may contribute to impaired response to IFN-γ in activated macrophages. This effect is negated by inhibition of NO synthase activity by concomitant treatment with l-NMMA, a competitive inhibitor of the enzyme (21). To investigate the possible involvement of NO in the defective Stat1 phosphorylation observed in CD8+ DCs from NOD females in response to IFN-γ, we exposed these cells to the cytokine in the presence of l-NMMA under conditions known to restore macrophage responsiveness to IFN-γ (21). Using an experimental setting as described above, we did not detect any effect of l-NMMA on Stat1 phosphorylation, IDO expression by Western blot analysis, or IDO functional activity as induced by IFN-γ (unpublished data). Peroxynitrite is a highly reactive oxidant resulting from the interaction of NO with superoxide, and is known to be produced in acutely diabetic NOD mice (6). Peroxynitrite may impair cell signaling via nitration of tyrosine residues (22). An inhibitor of NO synthase and scavenger of peroxynitrite (i.e., GED) prevents diabetes development in NOD mice (6). To investigate the possible production of peroxynitrites by DCs from NOD mice, we assayed IFN-γ–induced production of nitrotyrosine in CD8+ DCs from 4-wk-old female and male mice as well as control DBA/2 animals. NOD female mice were also assayed at 8 wk (Fig. 3). No nitrotyrosine formation was observed in cell supernatants or lysates of DCs unexposed to IFN-γ regardless of donor type (data not depicted). In contrast, IFN-γ induced high levels of peroxynitrite selectively in DCs from NOD female mice at 4 wk. In addition, GED prevented IFN-γ from inducing nitrotyrosine formation in these cells. In parallel, we examined the effect of GED on IFN-γ–induced Stat1 phosphorylation and IDO expression and function (Fig. 4). GED restored Stat1 phosphorylation and IDO protein expression (Fig. 4 A) as well as kynurenine production (Fig. 4 B) in CD8+ DCs from 4-wk-old NOD female mice treated with IFN-γ. Because NO is a precursor of peroxynitrite, the lack of activity of l-NMMA is not easily explained but could be related to the different relative potencies of l-NMMA and GED in inhibiting enzyme activity (21). Overall, these data demonstrate that CD8+ DCs from NOD female mice early in prediabetes produce peroxynitrite in response to IFN-γ. This oxidant species impairs the intracellular signal transduction pathways that lead to Stat1 phosphorylation and IDO expression in response to IFN-γ.


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)

Ability of GED to restore IFN-γ responsiveness in CD8+ DCs from early prediabetic NOD female mice. (A) GED was examined for ability to restore IDO expression and Stat1 phosphorylation in response to IFN-γ. CD8+ DCs from 4-wk-old NOD female mice were treated overnight with IFN-γ and assayed for IDO expression or Stat1 phosphorylation. One experiment representative of three. (B) GED was also studied for ability to restore kynurenine production by CD8+ DCs from the same donors. One experiment representative of three.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196078&req=5

fig4: Ability of GED to restore IFN-γ responsiveness in CD8+ DCs from early prediabetic NOD female mice. (A) GED was examined for ability to restore IDO expression and Stat1 phosphorylation in response to IFN-γ. CD8+ DCs from 4-wk-old NOD female mice were treated overnight with IFN-γ and assayed for IDO expression or Stat1 phosphorylation. One experiment representative of three. (B) GED was also studied for ability to restore kynurenine production by CD8+ DCs from the same donors. One experiment representative of three.
Mentions: In NOD mice, activated macrophages produce high amounts of nitric oxide (NO), which is thought to contribute to the destructive phase of insulitis (19, 20). There is evidence that tyrosine nitration of Stat1 by endogenous NO may contribute to impaired response to IFN-γ in activated macrophages. This effect is negated by inhibition of NO synthase activity by concomitant treatment with l-NMMA, a competitive inhibitor of the enzyme (21). To investigate the possible involvement of NO in the defective Stat1 phosphorylation observed in CD8+ DCs from NOD females in response to IFN-γ, we exposed these cells to the cytokine in the presence of l-NMMA under conditions known to restore macrophage responsiveness to IFN-γ (21). Using an experimental setting as described above, we did not detect any effect of l-NMMA on Stat1 phosphorylation, IDO expression by Western blot analysis, or IDO functional activity as induced by IFN-γ (unpublished data). Peroxynitrite is a highly reactive oxidant resulting from the interaction of NO with superoxide, and is known to be produced in acutely diabetic NOD mice (6). Peroxynitrite may impair cell signaling via nitration of tyrosine residues (22). An inhibitor of NO synthase and scavenger of peroxynitrite (i.e., GED) prevents diabetes development in NOD mice (6). To investigate the possible production of peroxynitrites by DCs from NOD mice, we assayed IFN-γ–induced production of nitrotyrosine in CD8+ DCs from 4-wk-old female and male mice as well as control DBA/2 animals. NOD female mice were also assayed at 8 wk (Fig. 3). No nitrotyrosine formation was observed in cell supernatants or lysates of DCs unexposed to IFN-γ regardless of donor type (data not depicted). In contrast, IFN-γ induced high levels of peroxynitrite selectively in DCs from NOD female mice at 4 wk. In addition, GED prevented IFN-γ from inducing nitrotyrosine formation in these cells. In parallel, we examined the effect of GED on IFN-γ–induced Stat1 phosphorylation and IDO expression and function (Fig. 4). GED restored Stat1 phosphorylation and IDO protein expression (Fig. 4 A) as well as kynurenine production (Fig. 4 B) in CD8+ DCs from 4-wk-old NOD female mice treated with IFN-γ. Because NO is a precursor of peroxynitrite, the lack of activity of l-NMMA is not easily explained but could be related to the different relative potencies of l-NMMA and GED in inhibiting enzyme activity (21). Overall, these data demonstrate that CD8+ DCs from NOD female mice early in prediabetes produce peroxynitrite in response to IFN-γ. This oxidant species impairs the intracellular signal transduction pathways that lead to Stat1 phosphorylation and IDO expression in response to IFN-γ.

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