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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.

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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.
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fig02: 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.

Mentions: It is well described that IDO+ NP-APC, such as mesenchymal stroma cells, fibroblasts and epithelial cells, as well as cells from different tumour lines, are capable of mediating inhibitory effects on T cells in an IDO-dependent fashion, when they are present as bystander cells during T-cell activation [35–37]. We assume that this immunoregulatory effect, mediated by NP-APC, does not have an incidental bystander function but rather is ubiquitously present as a part of the IFN-γ stimulation, resulting from inflammation. We have previously shown that immortalized human brain microvascular cells (HBMEC) are able to inhibit T-cell responses in an IDO-dependent fashion [38]. In addition, Figs 1–3 demonstrate that different tumour cells for example astrocytoma cells (86HG39), lung carcinoma cells (A549) or cervix carcinoma cells (HeLa) as well as native tissue cells (HFF) are, subsequent to IDO induction by IFN-γ, capable of inhibiting mitogen (PHA), superantigen (SEB), anti-CD3 and alloantigen-driven T-cell activation, when present as bystander cells during T-cell activation. These inhibitory effects could be blocked by the addition of tryptophan as well as by the addition of the IDO specific inhibitor 1-methyl-tryptophan. Further experiments indicated that the inhibitory effect mediated by NP-APC, present as bystander cells, was dependent on the number of cells present during T-cell activation. For example 3 x 104 86HG39 cells stimulated with IFN-γ for 72 hrs are able to inhibit the T-cell response by 70%, whereas 3 × 103 cells have no effect. In addition, we found that the IFN-γ pre-stimulation time was also critical in the mediation of this anti-proliferative effect: 3 × 104 86HG39 cells pre-treated with IFN-γ for only 24 hrs were unable to inhibit T-cell proliferation (Fig. 2). Using double culture systems, separated by a semi-permeable membrane, we found that this IDO-mediated anti-proliferative effect was independent of a cell–cell contact. Even culture supernatants, conditioned by IFN-γ activated cells, used as culture medium for subsequent T-cell activation, mediated this anti-proliferative effect (Fig. 3). As a control, supernatants from IFN-γ-activated Jurkat cells or from epstein-barr virus (EBV)-transformed B-cells, which do not express IDO activity, were used and did not influence T-cell proliferation (data not shown).


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)

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.
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Related In: Results  -  Collection

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fig02: 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.
Mentions: It is well described that IDO+ NP-APC, such as mesenchymal stroma cells, fibroblasts and epithelial cells, as well as cells from different tumour lines, are capable of mediating inhibitory effects on T cells in an IDO-dependent fashion, when they are present as bystander cells during T-cell activation [35–37]. We assume that this immunoregulatory effect, mediated by NP-APC, does not have an incidental bystander function but rather is ubiquitously present as a part of the IFN-γ stimulation, resulting from inflammation. We have previously shown that immortalized human brain microvascular cells (HBMEC) are able to inhibit T-cell responses in an IDO-dependent fashion [38]. In addition, Figs 1–3 demonstrate that different tumour cells for example astrocytoma cells (86HG39), lung carcinoma cells (A549) or cervix carcinoma cells (HeLa) as well as native tissue cells (HFF) are, subsequent to IDO induction by IFN-γ, capable of inhibiting mitogen (PHA), superantigen (SEB), anti-CD3 and alloantigen-driven T-cell activation, when present as bystander cells during T-cell activation. These inhibitory effects could be blocked by the addition of tryptophan as well as by the addition of the IDO specific inhibitor 1-methyl-tryptophan. Further experiments indicated that the inhibitory effect mediated by NP-APC, present as bystander cells, was dependent on the number of cells present during T-cell activation. For example 3 x 104 86HG39 cells stimulated with IFN-γ for 72 hrs are able to inhibit the T-cell response by 70%, whereas 3 × 103 cells have no effect. In addition, we found that the IFN-γ pre-stimulation time was also critical in the mediation of this anti-proliferative effect: 3 × 104 86HG39 cells pre-treated with IFN-γ for only 24 hrs were unable to inhibit T-cell proliferation (Fig. 2). Using double culture systems, separated by a semi-permeable membrane, we found that this IDO-mediated anti-proliferative effect was independent of a cell–cell contact. Even culture supernatants, conditioned by IFN-γ activated cells, used as culture medium for subsequent T-cell activation, mediated this anti-proliferative effect (Fig. 3). As a control, supernatants from IFN-γ-activated Jurkat cells or from epstein-barr virus (EBV)-transformed B-cells, which do not express IDO activity, were used and did not influence T-cell proliferation (data not shown).

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