Indoleamine 2,3-Dioxygenase Activity During Acute Toxoplasmosis and the Suppressed T Cell Proliferation in Mice

Abstract

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite and belongs to the phylum Apicomplexa. T. gondii is of medical and veterinary importance, because T. gondii causes the parasitic disease toxoplasmosis. In human cells, the interferon-gamma inducible indoleamine 2,3-dioxygenase 1 (IDO1) is an antimicrobial effector mechanism that degrades tryptophan to kynurenine and thus limits pathogen proliferation in vitro. Furthermore, IDO is described to have immunosuppressive properties, e.g., regulatory T cell differentiation and T cell suppression in humans and mice. However, there is only little known about the role of IDO1 in mice during acute toxoplasmosis. To shed further light on the role of mIDO1 in vivo, we have used a specifically adjusted experimental model. Therein, we infected mIDO1-deficient (IDO^-/-) C57BL/6 mice and appropriate wild-type (WT) control mice with a high dose of T. gondii ME49 tachyozoites (type II strain) via the intraperitoneal route and compared the phenotype of IDO^-/- and WT mice during acute toxoplasmosis. During murine T. gondii infection, we found mIDO1 mRNA and mIDO1 protein, as well as mIDO1-mediated tryptophan degradation in lungs of WT mice. IDO^-/- mice show no tryptophan degradation in the lung during infection. Even though T. gondii is tryptophan auxotroph and rapidly replicates during acute infection, the parasite load was similar in IDO^-/- mice compared to WT mice 7 days post-infection. IDO1 is described to have immunosuppressive properties, and since T cell suppression is observed during acute toxoplasmosis, we analyzed the possible involvement of mIDO1. Here, we did not find differences in the intensity of ex vivo mitogen stimulated T cell proliferation between WT and IDO^-/- mice. Concomitant nitric oxide synthase inhibition and interleukin-2 supplementation increased the T cell proliferation from both genotypes drastically, but not completely. In sum, we analyzed the involvement of mIDO1 during acute murine toxoplasmosis in our specifically adjusted experimental model and found a definite mIDO1 induction. Nevertheless, mIDO1 seems to be functional redundant as an antiparasitic defense mechanism during acute toxoplasmosis in mice. Furthermore, we suggest that the systemic T cell suppression observed during acute toxoplasmosis is influenced by nitric oxide activity and IL-2 deprivation.

Keywords: IDO; T cell suppression; Toxoplasma gondii; kynurenine; mouse.

Figure 1

Expression of mIDO1 in murine lung tissue during Toxoplasma gondii infection. Gene and protein expression in tissues from naïve or T. gondii ME49-infected wild-type (WT) and indoleamine 2,3-dioxygenase 1-deficient (IDO^−/−) mice. Western blot analysis shows murine IDO (mIDO) and β-actin protein in lung, liver, and brain tissue of naïve (–) and infected (+) WT and IDO^−/− mice (A). Expression of murine guanylate binding protein 2 (mGBP2), mIDO1, and mIDO2 in lung tissue homogenates of infected mice relative to their expression in naïve control samples (B). Data were normalized to the housekeeping gene β-actin and were represented as 2^−ΔΔCT (naïve vs. infected) in scattered dot plots and means ± standard deviation. The Student's t-test (unpaired, two-tailed) was used to determine statistical differences marked with asterisks (n.s., not significant; ***p ≤ 0.0001).

Figure 2

mIDO1 activity in murine lung tissue during Toxoplasma gondii infection. Serum and lung samples were collected from naïve or T. gondii ME49-infected wild-type (WT) and indoleamine 2,3-dioxygenase 1-deficient (IDO^−/−) mice. Free tryptophan and kynurenine was measured via high-performance liquid chromatography in serum (A) and lung tissue homogenate samples (B). Comparison of the T. gondii load (determined by B1-gene detection) in lungs of naïve or infected WT and IDO^−/− mice (C). Data are represented as scattered dot plots and means (±standard error of the mean for RT-PCR data). For the RT-PCR data, each dot represents the mean of duplicate measurements of a single mouse lung tissue sample. The Student's t-test (unpaired, two-tailed) was used to determine statistical differences marked with asterisks (n.s., not significant; *p ≤ 0.05, **p ≤ 0.001, and ***p ≤ 0.0001).

Figure 3

Mitogen-stimulated lymphocyte proliferation is impaired during acute toxoplasmosis in WT spleen and MLN-derived cells. Spleen (A) and mesenteric lymph nodes (MLN) (B) were isolated from naïve or T. gondii ME49-infected wild-type (WT) mice. Cell cultures were supplemented with the mitogen concanavalin A (ConA; 1 μg/ml), the class B phosphate linked cytosine, and guanine oligonucleotide ODN1826 (CpG B; 0.1 μM) to stimulate T cell and B cell proliferation ex vivo or were left untreated. Lymphocyte proliferation was determined with the ³H-thymidine method. Data were represented as means of triplicate measurements (n = 7–9) ± standard error of the mean. The Student's t-test (unpaired, two-tailed) was used to determine statistical differences marked with asterisks (n.s., not significant; *p ≤ 0.05, **p ≤ 0.001 and ***p ≤ 0.0001).

Figure 4

T cell proliferation is altered by IL-2 and NOS activity in splenocytes derived from WT and IDO^−/− mice. Splenocytes were isolated from naïve or T. gondii ME49-infected wild-type (WT) and indoleamine 2,3-dioxygenase 1-deficient (IDO^−/−) mice (WT naïve: n = 7; IDO^−/− naïve: n = 5; WT infected: n = 8; IDO^−/− infected: n = 10). Splenic T cell cultures were stimulated with the mitogen concanavalin A (ConA, 1 μg/ml) ex vivo. Supplementation of human interleukin-2 (IL-2, 5 ng/ml) and the nitric oxide synthase (NOS) inhibitor N^G-monomethyl-L-arginine (N^GMMA, 100 μg/ml) was done as indicated (A,B). Lymphocyte proliferation was determined with the ³H-thymidine method. (A). Nitrite accumulation in the supernatant of ex vivo cultured splenocytes was detected using the Griess reaction (B). Data were represented as means of triplicate measurements ± standard error of the mean. The Student's t-test (unpaired, two-tailed) was used to determine statistical differences marked with asterisks (n.s. = not significant; *p ≤ 0.05, **p ≤ 0.001, and ***p ≤ 0.0001).