The missing link between indoleamine 2,3-dioxygenase mediated antibacterial and immunoregulatory effects

Abstract

The interferon (IFN)-gamma-inducible tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO) has not only been recognized as a potent antimicrobial effector molecule for the last 25 years but was recently found also to have potent immunoregulatory properties. In this study, we provide evidence that both tryptophan starvation and production of toxic tryptophan metabolites are involved in the immunoregulation mediated by IDO, whereas tryptophan starvation seems to be the only antibacterial effector mechanism. A long-studied controversy in the IDO research field is the seemingly contradictory effect of IDO in the defence against infectious diseases. On the one hand, IFN-gamma-induced IDO activity mediates an antimicrobial effect, while at the same time IDO inhibits T-cell proliferation and IFN-gamma production. Here, we suggest that both effects, dependent on the threshold for tryptophan, cooperate in a reasonable coherence. We found that the minimum concentration of tryptophan required for bacterial growth is 10-40-fold higher than the minimum concentration necessary for T-cell activation. Therefore, we suggest that during the first phase of infection the IDO-mediated tryptophan depletion has a predominantly antimicrobial effect whereas in the next stage, and with ongoing tryptophan degradation, the minimum threshold concentration of tryptophan for T-cell activation is undercut, resulting in an inhibition of T-cell growth and subsequent IDO activation.

Figure 1

IDO⁺ tumour cells are capable of inhibiting mitogen-driven T-cell proliferation: Mitomycin-treated 86HG39 cells (A) or irradiated A549 cells (B) were incubated with or without IFN-γ (500 U/ml). After 3 days, 1.5 × 10⁵ PBL were added and stimulated with anti-CD3 antibodies, phytohemagglu-tinin A or staphylococcal enterotoxin B for additional 3 days in the absence or presence of tryptophan (100 μg/ml). T-cell proliferation was determined by ³H-thymidine incorporation after 3 days. Data are given as mean ± S.E.M. of two independent experiments, each done in triplicate. Asterisks indicate significant inhibition of T-cell responses (P < 0.005).

Figure 2

Time and dose dependency of IDO-mediated inhibition of T-cell growth by 86HG39 cells. Mitomycin-treated 86HG39 cells (3 × 10⁴/well) were incubated for 24, 48 or 72 hrs in the presence or absence of IFN-γ (500 U/ml). After 3 days, 1 × 10⁵ alloreactive T cells were added for additional 3 days in absence or presence of tryptophan (100 μg/ml). T-cell proliferation was determined by ³H-thymidine incorporation after 3 days. Data are given as mean ± S.E.M. of two independent experiments, each done in triplicate (A). The identical read-out system was used in part B, but different amounts of 86HG39 cells/well were used and stimulated for 3 days.

Figure 3

Inhibition of T-cell growth in media, conditioned by IDO⁺ cells. 1.25 × 10⁶ HeLa cells (A) or human foreskin fibroblasts (B) were incubated in the presence of IFN-γ (500 U/ml) or of IFN-γ and 1-methyl-L-tryptophan (1-MT) for 3 days. Thereafter, supernatants were collected and used as culture medium for PBL (1 x 10⁵/well) and stimulated with anti-CD3 antibodies either in presence or absence of tryptophan (100 μg/ml). T-cell proliferation was determined by ³H-thymidine incorporation after 3 days and data are given as mean ± S.E.M. of two independent experiments, each done in triplicate. Asterisks indicate significant inhibition of T-cell responses (P < 0.001).

Figure 4

Differences in the tryptophan antagonism of antibacterial and anti-proliferative effects mediated by IDO⁺ 86HG39 cells. A total of 5 x 10⁶ 86HG39 cells were incubated with or without IFN-7 (500 U/ml) in the presence of different concentrations of additional tryptophan (0, 25, 50 or 100 μg/ml). After 3 days of incubation, supernatants were harvested and the kynurenine content was measured as described in materials and methods (A). In addition, Staphylococcus aureus (10-50 CFU/well) was added to the supernatants in the absence (white bars) or presence (grey bars) of tryptophan (100 μg/ml). Bacterial growth was determined photometrically after 24 hrs (B). Furthermore, cell supernatants were used as culture medium for PBL (1 × 10⁵/well, suspended in 10 μl of trypto-phan-free RPMI 1640 medium), stimulated with anti-CD3 antibodies either in presence (grey bars) or absence (white bars) of tryptophan (100 μg/ml) (C). Data were given as mean ± S.E.M. of three independent experiments, each done in triplicate. Asterisks indicate significant inhibition of bacterial growth or of T-cell responses (P < 0.001).

Figure 5

Different effects of kynurenine on the proliferation of T cells and bacteria. Staphylococcal growth in presence of different concentrations of kynurenine was analysed photometrically (A). Anti-CD3-driven T-cell activation was analysed in absence or presence of different concentrations of kynurenine (B). In both experiments, a part of the cultures was supplemented with tryptophan (100 μg/ml). Data are given as mean ± S.E.M. of three independent experiments, each done in triplicate.

Figure 6

Differences in the tryptophan requirement between T cells and bacteria. Supernatants harvested from A459 cells, incubated with or without IFN-g (1000 U/ml), were used as culture medium. Anti-CD3-driven T-cell proliferation in conditioned medium in the presence or absence of different concentrations of supplemental tryptophan (A). Staphylococcal growth in conditioned medium in the presence or absence of different concentrations of supplemental tryptophan was determined photometrically (B) or using the cultivation method (C). Data are given as mean ± S.E.M. of 18 independent experiments, each done in triplicate. A dotted line indicates half maximal responses. The paired t-test indicates a significant lower tryptophan requirement for T cells in comparison to bacteria for all tryptophan concentrations below 0.8 mg/ml (P= 0.0014).

Figure 7

Determination of the tryptophan requirement of different bacteria. Four different strains of Staphylococcus aureus (A) or four different strains of enterococci (B) were cultured in conditioned medium from IFN-g (500 U/ml) stimulated A549 cells in absence or presence of different concentrations of tryptophan. Data are shown as mean ± S.E.M. of four independent experiments, each done in triplicate. A dotted line indicates half maximal proliferation.