Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and kynurenine formation during in vivo infection with Toxoplasma gondii: induction by endogenous gamma interferon and requirement of interferon regulatory factor 1

Abstract

The induction of indoleamine 2,3-dioxygenase (INDO) expression and the tryptophan (Trp)-kynurenine (Kyn) metabolic pathway during in vivo infection with Toxoplasma gondii was investigated. Decreased levels of Trp and increased formation of Kyn were observed in the lungs, brain, and serum from mice infected with T. gondii. Maximal INDO mRNA expression and enzyme activity were detected in the lungs at 10 to 20 days postinfection. Further, the induction of INDO mRNA expression, Trp degradation and Kyn formation were completely absent in tissues from mice deficient in IFN-gamma (IFN-gamma(-/-)) or IFN regulatory factor -1 (IRF-1(-/-)). These findings indicate the important role of endogenous IFN-gamma and IRF-1 in the in vivo induction of the Trp-Kyn metabolic pathway during acute infection with T. gondii. In contrast, expression of INDO mRNA and its activity was preserved in the tissues of TNF-receptor p55- or inducible nitric oxide synthase-deficient mice infected with T. gondii. Together with the results showing the extreme susceptibility of the IFN-gamma(-/-) and the IRF-1(-/-) mice to infection with T. gondii, our results indicate a possible involvement of INDO and Trp degradation in host resistance to early infection with this parasite.

FIG. 1.

Mortality rates of ME-49 T. gondii-infected TNFRp55^−/−, iNOS^−/−, IFN-γ^−/−, IRF-1^−/− mice and their respective C57BL/6 and 129 WT mice. IFN-γ^−/− (▾) and IRF-1^−/− (○) mice were significantly more susceptible to toxoplasmosis than were TNFRp55^−/− (•), iNOS^−/− (▴), and WT mice C57BL/6 (▪) and 129 (□) (P < 0.05). TNFRp55^−/− and iNOS^−/− were more susceptible than their WT counterparts (P < 0.001). The mortality rate for 10 mice from each group was determined. The results from one of two experiments, which yielded similar results, are shown.

FIG. 2.

T. gondii-specific immunoperoxidase staining in tissues from IFN-γ^−/− and iNOS^−/− mice infected with the ME-49 strain of T. gondii. (A) A large number of individual tachyzoites are shown in liver tissue from IFN-γ^−/− mice at 7 days postinfection. (B) Cyst like structures are shown in CNS from iNOS^−/− mice at 21 days postinfection. Original magnification, ×100.

FIG. 3.

PCR analysis of the T. gondii 35-copy B1 gene in pulmonary and hepatic tissues from iNOS^−/−, IFN-γ^−/−, and C57BL/6 mice. (A) Sensitivity determinations of the 35-copy B1 gene PCR by using different concentrations of DNA extracted from tachyzoites from the ME-49 strain of T. gondii. (B) The amplification product of B1 gene in DNA samples extracted from lungs and liver from WT, iNOS^−/−, and IFN-γ^−/− mice at 5, 7 to 9, 14, and 21 days after infection with T. gondii. Each lane corresponds to the PCR product obtained from the lung or liver of a single mouse. (C) Correlation between densitometric analysis from a semiquantitative PCR and morphometric assays from immunoperoxidase staining in tissues from iNOS^−/−, IFN-γ^−/−, and C57BL/6 mice.

FIG. 4.

Kinetics of tryptophan and kynurenine concentrations in serum and tissue samples from C57BL/6 mice infected with T. gondii. (Left) Total free Trp (▪) and Kyn (□) were quantified by HPLC. Values presented are means and standard deviations obtained with three mice per group. One and two asterisks indicate that Trp (p < 0.05) or Kyn (p < 0.01) concentrations are significantly different from those obtained from noninfected mice. (Right) Chromatograms showing total free Trp and Kyn in pulmonary tissue from noninfected (top) and T. gondii-infected (bottom) mice. The detector UV light was used at 280 and 365 nm to measure Trp and Kyn levels, respectively. The retention times of Trp and Kyn are indicated by the arrows. Similar results were obtained in two different experiments.

FIG. 5.

Expression of IFN-γ, iNOS, and INDO mRNA genes in the lungs from mice infected with T. gondii ME-49. Lungs from C57BL/6 (top), 129 and IRF-1^−/− (middle), and IFN-γ^−/−, TNFRp55^−/−, and iNOS^−/− (bottom) mice were harvested at different times postinfection, total RNA was extracted, and gene expression was evaluated by RT-PCR. The amplification products for IFN-γ (237 pb), iNOS (832 pb), INDO (1,133 pb) and HPRT (160 pb) cDNAs were visualized in a silver-stained polyacrylamide gel. Each lane corresponds to the RT-PCR product obtained from the lungs of a single mouse. Similar results were obtained in two different experiments.

FIG. 6.

Tryptophan and kynurenine concentrations in serum, lung, and CNS samples from TNFRp55^−/−, iNOS^−/−, IFN-γ^−/−, and IRF-1^−/− mice on days 0, 5, and 8 after infection with T. gondii. Total free Trp (▪) and kyn (□) were quantified by HPLC. Values presented are means and standard deviations obtained from three mice per group. One and two asterisks indicate that Trp (p < 0.01) or Kyn (p < 0.01) concentrations are significantly different from those obtained from non-infected mice. Similar results were obtained in two different experiments.