L-tryptophan-L-kynurenine pathway metabolism accelerated by Toxoplasma gondii infection is abolished in gamma interferon-gene-deficient mice: cross-regulation between inducible nitric oxide synthase and indoleamine-2,3-dioxygenase

Abstract

L-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) might have an important role in gamma interferon (IFN-gamma)-induced antimicrobial effects. In the present study, the effects of Toxoplasma gondii infection on IDO were investigated by using wild-type and IFN-gamma-gene-deficient (knockout) (IFN-gamma KO) mice. In wild-type C57BL/6J mice, enzyme activities and mRNA levels for IDO in both lungs and brain were markedly increased and lung L-tryptophan concentrations were dramatically decreased following T. gondii infection. In contrast, these metabolic changes did not occur in T. gondii-infected IFN-gamma KO mice or in uninfected IFN-gamma KO mice. The levels of inducible nitric oxide synthase (iNOS) induction in infected IFN-gamma KO mice were high in lungs and low in brain compared to those in infected wild-type mice. The extent of increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) induced in lungs and brain by T. gondii infection was significantly enhanced in IFN-gamma KO mice compared to wild-type mice on day 7 postinfection. Treatment with N-nitro-L-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain but not in lungs and of plasma L-kynurenine after T. gondii infection. This in vivo study provides evidence that L-tryptophan depletion caused by T. gondii is directly mediated by IFN-gamma in the lungs, where iNOS is not induced by IFN-gamma. This study suggests that there is an antitoxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of the main antiparasite effector mechanisms for iNOS and/or IDO may vary among tissues.

FIG. 1.

Accumulation of plasma l-KYN (A) and marked induction of IDO activity in lungs (B) and brain (C) by T. gondii infection in wild-type mice. Note the logarithmic scale of the ordinate. Samples were analyzed on days 0, 4, 7, 14, and 28 following intraperitoneal injection of 20 cysts of T. gondii. Each point represents the mean and SEM for results from three to six samples. In a preliminary study (not shown), there were no significant changes in parameters measured in wild-type mice treated with brain homogenate without T. gondii infection at any time examined. ∗, P < 0.05. ∗∗, P < 0.005.

FIG. 2.

Induction of IDO mRNA expression in lungs (A) and brain (B) following T. gondii infection in wild-type mice. Total RNA was isolated from the lungs and brain of wild-type mice on the indicated days following intraperitoneal injection of T. gondii and used for RT-PCR. Each point represents the mean and SEM for results from three to six samples. ∗∗, P < 0.005 (for comparisons with controls).

FIG. 3.

Effects of IFN-γ gene deficiency on the degradation of l-TRP and the accumulation of l-KYN in lungs and brain after T. gondii infection. The concentrations of l-TRP (A) and l-KYN (B) in supernatants of homogenates of lungs and brain from wild-type and IFN-γ KO mice on days 0 and 7 following intraperitoneal injection of T. gondii were measured by using HPLC. Each bar represents the mean and SEM for results from four to eight samples. ∗∗, P < 0.005 (for comparisons with day 0 postinfection). It is especially noteworthy that IDO activity in the lungs was dramatically increased (more than 100-fold) by toxoplasma infection. Surprisingly, the l-TRP concentration in the lungs was completely depleted.

FIG. 4.

Effects of IFN-γ gene deficiency on the induction of IDO and iNOS and tachyzoite SAG2 mRNA expression by T. gondii infection. The expression of IDO mRNA (A and B), iNOS mRNA (C and D), and SAG2 mRNA (E) was analyzed by using RT-PCR with total RNA from the lungs and brain of wild-type and IFN-γ KO mice on days 0 and 7 following intraperitoneal injection of T. gondii. Each bar represents the mean and SEM for results from three to six samples. ∗, P < 0.05 (for comparisons with day 0 postinfection). ∗∗, P < 0.005 (for comparisons with day 0 postinfection). (F) Representative gel photograph.

FIG. 5.

Effects of an iNOS inhibitor on the plasma l-KYN and nitrate concentrations and tachyzoite SAG2 mRNA expression in T. gondii infection. Plasma l-KYN (A) and nitrate (NOx) (B) concentrations and tachyzoite SAG2 mRNA expression (C) in uninfected and infected mice treated with saline (Control) or l-NAME on day 7 following intraperitoneal injection of T. gondii were examined. Each bar represents the mean and SEM for results from four to six samples. ∗, P < 0.05 (for comparisons with controls). ∗∗, P < 0.005 (for comparisons with controls).