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

Müller A, Heseler K, Schmidt SK, Spekker K, Mackenzie CR, Däubener W - J. Cell. Mol. Med. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-Universität Düsseldorf, Germany.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4496108&req=5

fig07: 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.

Mentions: We have recognized that IDO+ NP-APCs are capable of inhibiting both T-cell proliferation and bacterial growth. These two IDO-mediated effects seem to be contradictory in terms of a unifying immune defence hypothesis. To analyse the relationship between IDO-mediated antimicrobial and immunoregulatory effects in more detail, we aimed to define the threshold concentration of tryptophan, necessary to mediate both effects. Therefore, we once again used supernatants harvested from IDO+ cells to determine bacterial growth and T-cell growth and supplemented these conditioned media with different amounts of tryptophan. Figure 6B shows the bacterial growth, measured by determining optical density. These data indicate that bacterial growth is blocked in the supernatant of IFN-γ-stimulated cells and that this antibacterial effect could be restored by the addition of tryptophan. A 50% inhibition of bacterial growth was found at a tryptophan concentration of about 0.38 μg/ml (1.9 μM). The same supernatants were also used to culture T cells and the data in Fig. 6A indicate that T-cell proliferation in medium, conditioned by IFN-7 activated A549 cells, is reduced by about 70%. A half-maximal inhibition of T-cell growth was achieved at a tryptophan concentration of about 0.02 ixg/ml (0.1 jjlM) (Fig. 6). Hence, we found a 19-fold difference in the tryptophan need for T cells compared to bacteria. In order to analyse this difference in more detail, we determined CFU as a second and a more sensitive readout system, to quantify bacterial growth in four additional experiments. We noticed that bacterial growth in conditioned medium, harvested from IFN-γ activated cells, was dramatically reduced from 1 × 109 CFU/ml in the supernatant of untreated cells to 1 CFU/ml in the supernatant of IFN-γ activated cells (Fig. 6C). A half-maximal reduction of bacterial growth, using the CFU method, was ascertained at a tryptophan concentration of 0.82 μg/ml (4.1 μM), which is 41-fold higher than the threshold concentration for T cells. All experiments shown in Fig. 6 were done with one strain of Staphylococcus aureus. To ensure that the comparatively high threshold concentration is consistent with other strains we also analysed four additional Staphylococcus aureus isolates and obtained comparable results. Here, we detected again a 50% inhibition of bacterial growth at a tryptophan concentration of about 0.3 μg/ml. Furthermore, four strains of enterococci were tested and showed a 50% inhibition of bacterial growth at a tryptophan concentration of 0.2 μg/ml (1.0 μM) (Fig. 7). Altogether, these data indicate that the tryptophan need of bacteria is higher than the tryptophan need of T cells. This led us to the conclusion that a comparably lesser tryptophan degradation mediates an antibacterial effect whereas a 10–40-fold stronger tryptophan depletion is necessary to mediate an immunoregulatory effect.


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

Müller A, Heseler K, Schmidt SK, Spekker K, Mackenzie CR, Däubener W - J. Cell. Mol. Med. (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496108&req=5

fig07: 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.
Mentions: We have recognized that IDO+ NP-APCs are capable of inhibiting both T-cell proliferation and bacterial growth. These two IDO-mediated effects seem to be contradictory in terms of a unifying immune defence hypothesis. To analyse the relationship between IDO-mediated antimicrobial and immunoregulatory effects in more detail, we aimed to define the threshold concentration of tryptophan, necessary to mediate both effects. Therefore, we once again used supernatants harvested from IDO+ cells to determine bacterial growth and T-cell growth and supplemented these conditioned media with different amounts of tryptophan. Figure 6B shows the bacterial growth, measured by determining optical density. These data indicate that bacterial growth is blocked in the supernatant of IFN-γ-stimulated cells and that this antibacterial effect could be restored by the addition of tryptophan. A 50% inhibition of bacterial growth was found at a tryptophan concentration of about 0.38 μg/ml (1.9 μM). The same supernatants were also used to culture T cells and the data in Fig. 6A indicate that T-cell proliferation in medium, conditioned by IFN-7 activated A549 cells, is reduced by about 70%. A half-maximal inhibition of T-cell growth was achieved at a tryptophan concentration of about 0.02 ixg/ml (0.1 jjlM) (Fig. 6). Hence, we found a 19-fold difference in the tryptophan need for T cells compared to bacteria. In order to analyse this difference in more detail, we determined CFU as a second and a more sensitive readout system, to quantify bacterial growth in four additional experiments. We noticed that bacterial growth in conditioned medium, harvested from IFN-γ activated cells, was dramatically reduced from 1 × 109 CFU/ml in the supernatant of untreated cells to 1 CFU/ml in the supernatant of IFN-γ activated cells (Fig. 6C). A half-maximal reduction of bacterial growth, using the CFU method, was ascertained at a tryptophan concentration of 0.82 μg/ml (4.1 μM), which is 41-fold higher than the threshold concentration for T cells. All experiments shown in Fig. 6 were done with one strain of Staphylococcus aureus. To ensure that the comparatively high threshold concentration is consistent with other strains we also analysed four additional Staphylococcus aureus isolates and obtained comparable results. Here, we detected again a 50% inhibition of bacterial growth at a tryptophan concentration of about 0.3 μg/ml. Furthermore, four strains of enterococci were tested and showed a 50% inhibition of bacterial growth at a tryptophan concentration of 0.2 μg/ml (1.0 μM) (Fig. 7). Altogether, these data indicate that the tryptophan need of bacteria is higher than the tryptophan need of T cells. This led us to the conclusion that a comparably lesser tryptophan degradation mediates an antibacterial effect whereas a 10–40-fold stronger tryptophan depletion is necessary to mediate an immunoregulatory effect.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-Universität Düsseldorf, Germany.

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.

Show MeSH
Related in: MedlinePlus