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Cited by 105Tuberculosis Research and EpidemiologyImmune responses and vaccinationsInfectious Diseases and Tuberculosis

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M. tuberculosis Induces Potent Activation of IDO-1, but This Is Not Essential for the Immunological Control of Infection

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Blumenthal, Antje, Nagalingam, Gayathri, Huch, Jennifer H., Walker, Lara, Guillemin, Gilles J., Smythe, George A., Ehrt, Sabine, Britton, Warwick J., Saunders, Bernadette M.·2012·PLoS ONE
DOI 10.1371/journal.pone.0037314PMC PMC3359358
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Abstract

Indoleamine 2,3-dioxygenesae-1 (IDO-1) catalyses the initial, rate-limiting step in tryptophan metabolism, thereby regulating tryptophan availability and the formation of downstream metabolites, including picolinic and quinolinic acid. We found that Mycobacterium tuberculosis infection induced marked upregulation of IDO-1 expression in both human and murine macrophages in vitro and in the lungs of mice following aerosol challenge with M. tuberculosis. The absence of IDO-1 in dendritic cells enhanced the activation of mycobacteria-specific T cells in vitro. Interestingly, IDO-1-deficiency during M. tuberculosis infection in mice was not associated with altered mycobacteria-specific T cell responses in vivo. The bacterial burden of infected organs, pulmonary inflammatory responses, and survival were also comparable in M. tuberculosis-infected IDO-1 deficient and wild type animals. Tryptophan is metabolised into either picolinic acid or quinolinic acid, but only picolinic acid inhibited the growth of M. tuberculosis in vitro. By contrast macrophages infected with pathogenic mycobacteria, produced quinolinic, rather than picolinic acid, which did not reduce M. tuberculosis growth in vitro. Therefore, although M. tuberculosis induces robust expression of IDO-1 and activation of tryptophan metabolism, IDO-1-deficiency fails to impact on the immune control and the outcome of the infection in the mouse model of tuberculosis.

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Figure 1 M. tuberculosis infection increases IDO-1 expression in human and murine macrophages. (A) Microarray results of BAL cells (>90% macrophages) infected with M. tuberculosis (MOI 3–5) for 24 h compared to uninfected cells. Increased mRNA expression levels for Indoleamine 2,3-dioxygenase (IDO-1), Kynurenine 3-monooxygenase (KYN-OX), and L-Kynurenine hydrolase (KYN-HYD) are depicted as fold induction upon M. tuberculosis infection compared to uninfected control cells. Data are means +/− standard error of comparisons of microarray data obtained from four different individuals. (B) Murine bone marrow-derived macrophages were left untreated (1), infected with M. tuberculosis (MOI 5) (2) and stimulated with IFN-γ (100 U/ml) (3). Co-stimulation with both M. tuberculosis and IFN-γ was either performed simultaneously (4) or cells were pre-treated with IFN-γ for 24 h and then infected with M. tuberculosis for 24 h (5) before RNA isolation. IDO-1 mRNA expression and beta-2-microglobulin (control) were detected at 4, 24, and 48 h by RT-PCR. (6) H2O. Data are representative of two independent experiments.
Figure 1 M. tuberculosis infection increases IDO-1 expression in human and murine macrophages. (A) Microarray results of BAL cells (>90% macrophages) infected with M. tuberculosis (MOI 3–5) for 24 h compared to uninfected cells. Increased mRNA expression levels for Indoleamine 2,3-dioxygenase (IDO-1), Kynurenine 3-monooxygenase (KYN-OX), and L-Kynurenine hydrolase (KYN-HYD) are depicted as fold induction upon M. tuberculosis infection compared to uninfected control cells. Data are means +/− standard error of comparisons of microarray data obtained from four different individuals. (B) Murine bone marrow-derived macrophages were left untreated (1), infected with M. tuberculosis (MOI 5) (2) and stimulated with IFN-γ (100 U/ml) (3). Co-stimulation with both M. tuberculosis and IFN-γ was either performed simultaneously (4) or cells were pre-treated with IFN-γ for 24 h and then infected with M. tuberculosis for 24 h (5) before RNA isolation. IDO-1 mRNA expression and beta-2-microglobulin (control) were detected at 4, 24, and 48 h by RT-PCR. (6) H2O. Data are representative of two independent experiments.CC BY · PubMed Central
Figure 2 Mycobacteria and IFN-γ activate tryptophan-kynurenine metabolism in macrophages. (A) Human bronco-alveolar lavage cells were infected with M. tuberculosis (MOI 3) and stimulated with IFN-γ (100 and 500 U/ml) for 48 h. The concentration of quinolinic acid in cell culture supernatants was detected by electron-capture negative-ion gas chromatography-mass spectrometry. Data points are means +/− SD of triplicate wells representative of two independently performed experiments. Unpaired t test was used to compare raw data of three independent wells. (***P = 0.0003, **P<0.01, all other comparisons showed no significant difference) (B) Quinolinic and (C) picolinic acid concentrations in cell culture supernatants of human monocyte-derived macrophages stimulated overnight with IFN-γ (100 U/ml) before infection for 24, 48, and 72 h with M. bovis BCG (MOI 5). Data points represent measurements obtained for cells from two individuals.
Figure 2 Mycobacteria and IFN-γ activate tryptophan-kynurenine metabolism in macrophages. (A) Human bronco-alveolar lavage cells were infected with M. tuberculosis (MOI 3) and stimulated with IFN-γ (100 and 500 U/ml) for 48 h. The concentration of quinolinic acid in cell culture supernatants was detected by electron-capture negative-ion gas chromatography-mass spectrometry. Data points are means +/− SD of triplicate wells representative of two independently performed experiments. Unpaired t test was used to compare raw data of three independent wells. (***P = 0.0003, **P<0.01, all other comparisons showed no significant difference) (B) Quinolinic and (C) picolinic acid concentrations in cell culture supernatants of human monocyte-derived macrophages stimulated overnight with IFN-γ (100 U/ml) before infection for 24, 48, and 72 h with M. bovis BCG (MOI 5). Data points represent measurements obtained for cells from two individuals.CC BY · PubMed Central
Figure 3 IDO-1 regulates CD4 T cell responses to mycobacterial antigen in vitro. Bone marrow-derived DCs were cultured for 4 days in vitro before stimulation for 2 h with Ag85 peptide P25. DCs were overlaid with CFSE labelled CD4+ T cells from P25-specific T cell receptor transgenic mice. Proliferation of T cells was determined by flow cytometry. DCs from WT and IDO-1−/− mice were compared in their ability to induce proliferation of P25-specific T cells after 2 days (A, D) and 5 days (B, E). WT DCs were treated with 1 Methyl-DL-tryptophan before stimulation with P25 peptide. P25-specific T cell proliferation was determined after 5 days (C, F). (A-C) Fluorescence intensities of representative FACS plots are depicted. Grey- IDO-1−/−/1-Methyl-DL-tryptophan, Black – WT/untreated control. The percentages of cells per division were calculated (D-F). Data represent the mean +/− SD of triplicate cultures from one of three representative experiments. The differences between groups were analysed by analysis of variance, *P<0.001.
Figure 3 IDO-1 regulates CD4 T cell responses to mycobacterial antigen in vitro. Bone marrow-derived DCs were cultured for 4 days in vitro before stimulation for 2 h with Ag85 peptide P25. DCs were overlaid with CFSE labelled CD4+ T cells from P25-specific T cell receptor transgenic mice. Proliferation of T cells was determined by flow cytometry. DCs from WT and IDO-1−/− mice were compared in their ability to induce proliferation of P25-specific T cells after 2 days (A, D) and 5 days (B, E). WT DCs were treated with 1 Methyl-DL-tryptophan before stimulation with P25 peptide. P25-specific T cell proliferation was determined after 5 days (C, F). (A-C) Fluorescence intensities of representative FACS plots are depicted. Grey- IDO-1−/−/1-Methyl-DL-tryptophan, Black – WT/untreated control. The percentages of cells per division were calculated (D-F). Data represent the mean +/− SD of triplicate cultures from one of three representative experiments. The differences between groups were analysed by analysis of variance, *P<0.001.CC BY · PubMed Central
Figure 4 Normal cellular influx and the generation of antigen-specific T cells in the absence of IDO-1 in vivo. WT and IDO-1−/− mice were infected with M. tuberculosis (100 CFU). Single cell suspensions were prepared from infected lungs and analyzed by flow cytometry for (A) CD4+, (B) CD8+ cells, and the percentages of these cells with an activated CD44hi/CD62lo phenotype ((C) CD4+, (D) CD8+). (E) Numbers of IFN-γ producing cells determined by ELISpot for lung cells cultured at 1×105 cells/well with M. tuberculosis CFP overnight. Data represent the mean +/− SD of 5 mice from one representative of two experiments.
Figure 4 Normal cellular influx and the generation of antigen-specific T cells in the absence of IDO-1 in vivo. WT and IDO-1−/− mice were infected with M. tuberculosis (100 CFU). Single cell suspensions were prepared from infected lungs and analyzed by flow cytometry for (A) CD4+, (B) CD8+ cells, and the percentages of these cells with an activated CD44hi/CD62lo phenotype ((C) CD4+, (D) CD8+). (E) Numbers of IFN-γ producing cells determined by ELISpot for lung cells cultured at 1×105 cells/well with M. tuberculosis CFP overnight. Data represent the mean +/− SD of 5 mice from one representative of two experiments.CC BY · PubMed Central
Figure 5 Control of M. tuberculosis infection by macrophages in the absence of IDO-1 activity. (A) Bone marrow-derived macrophages of WT and IDO-1−/− mice were infected with M. tuberculosis (MOI 1) in the presence of IFN-γ (100 U/ml). Intracellular bacterial numbers were determined at the indicated time points. Data points are means +/− SD of triplicate wells of one representative of 3 independent experiments. (B) Human BAL cells were stimulated with 100 U/ml IFN-γ and infected with M. tuberculosis (MOI 1) in the presence or absence of 1-Methyl-DL-Tryptophan (250 µM). Bacterial counts in cell lysates were determined at the times indicated. Data points are means +/− SD of triplicate wells of one representative of 6 independent experiments.
Figure 5 Control of M. tuberculosis infection by macrophages in the absence of IDO-1 activity. (A) Bone marrow-derived macrophages of WT and IDO-1−/− mice were infected with M. tuberculosis (MOI 1) in the presence of IFN-γ (100 U/ml). Intracellular bacterial numbers were determined at the indicated time points. Data points are means +/− SD of triplicate wells of one representative of 3 independent experiments. (B) Human BAL cells were stimulated with 100 U/ml IFN-γ and infected with M. tuberculosis (MOI 1) in the presence or absence of 1-Methyl-DL-Tryptophan (250 µM). Bacterial counts in cell lysates were determined at the times indicated. Data points are means +/− SD of triplicate wells of one representative of 6 independent experiments.CC BY · PubMed Central
Figure 6 IDO-1 is dispensable for the control of M. tuberculosis infection in vivo. WT and IDO-1−/− mice were infected with M. tuberculosis (100 CFU). IDO-1 expression in the lungs of uninfected WT mice (A), and WT (B) and IDO-1−/− (C) mice at 28 days post infection. Lungs from uninfected IDO-1−/− mice showed no IDO-1-positive cells (data not shown). IDO-1-expression in lungs of WT mice was primarily associated with inflammatory lesions within large monocytic cells (B), while IDO-1−/− mice show no specific staining (C). One representative section from 1 of 5 mice per group. Bacterial loads (D) in infected lungs were determined at the time points indicated. Data represent the means +/− SD of 5 mice per group from one of two independent experiments. Lung inflammation (E) as percentage of the lung area with inflammatory cell infiltration from WT and IDO-1−/− mice after subtracting the background cellularity in normal lung. To assess survival (F), infected mice were monitored daily and euthanized when showing signs of ill health. Data represent the time to euthanasia of 6 mice per group.
Figure 6 IDO-1 is dispensable for the control of M. tuberculosis infection in vivo. WT and IDO-1−/− mice were infected with M. tuberculosis (100 CFU). IDO-1 expression in the lungs of uninfected WT mice (A), and WT (B) and IDO-1−/− (C) mice at 28 days post infection. Lungs from uninfected IDO-1−/− mice showed no IDO-1-positive cells (data not shown). IDO-1-expression in lungs of WT mice was primarily associated with inflammatory lesions within large monocytic cells (B), while IDO-1−/− mice show no specific staining (C). One representative section from 1 of 5 mice per group. Bacterial loads (D) in infected lungs were determined at the time points indicated. Data represent the means +/− SD of 5 mice per group from one of two independent experiments. Lung inflammation (E) as percentage of the lung area with inflammatory cell infiltration from WT and IDO-1−/− mice after subtracting the background cellularity in normal lung. To assess survival (F), infected mice were monitored daily and euthanized when showing signs of ill health. Data represent the time to euthanasia of 6 mice per group.CC BY · PubMed Central
Figure 7 Inhibition of mycobacterial growth in vitro by picolinic acid. (A) M. tuberculosis was grown in 7H9 liquid medium in the presence of picolinic acid (100 - 1000 µM). Optical density of cultures was measured. Data are the mean +/− SD of four independent wells per condition and are representative of 2–4 independent experiments. M. tuberculosis CFU were determined upon exposure to picolinic acid (0.1 and 1 mM; HEPES and MES as buffer controls) in 7H9 liquid medium at pH 6.6 (B) and 5.5 (C). Data represent means +/− SD of triplicate wells. Similar pH-dependent effects of picolinic acid on M. tuberculosis survival were observed in three independent experiments.
Figure 7 Inhibition of mycobacterial growth in vitro by picolinic acid. (A) M. tuberculosis was grown in 7H9 liquid medium in the presence of picolinic acid (100 - 1000 µM). Optical density of cultures was measured. Data are the mean +/− SD of four independent wells per condition and are representative of 2–4 independent experiments. M. tuberculosis CFU were determined upon exposure to picolinic acid (0.1 and 1 mM; HEPES and MES as buffer controls) in 7H9 liquid medium at pH 6.6 (B) and 5.5 (C). Data represent means +/− SD of triplicate wells. Similar pH-dependent effects of picolinic acid on M. tuberculosis survival were observed in three independent experiments.CC BY · PubMed Central

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Authors
Blumenthal, A., Nagalingam, G., Huch, J. H., Walker, L., Guillemin, G. J., Smythe, G. A., Ehrt, S., Britton, W. J., & Saunders, B. M.
Affiliations
  • The University of Queensland (AU)
  • Centenary Institute (AU)
  • UNSW Sydney (AU)
  • Cornell University (US)
  • The University of Sydney (AU)
Corresponding author
  • Blumenthal, Antje
  • Saunders, Bernadette M.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-05-23
Volume
7
Issue
5
Pages
e37314
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0037314
PMC
PMC3359358
OpenAlex
W2025969665
Cited by
105
References
55
Citations by year
2026: 4 · 2025: 3 · 2024: 7 · 2023: 7 · 2022: 6 · 2021: 10 · 2020: 14 · 2019: 7 · 2018: 10 · 2017: 7 · 2016: 6 · 2015: 11 · 2014: 6 · 2013: 5 · 2012: 2
FWCI
5.30
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • UTS ePRESS (University of Technology Sydney) (submittedVersion)
Funders
  • National Institutes of Health
  • Medical Research Council
  • National Health and Medical Research Council
  • National Heart, Lung, and Blood Institute (R01HL068525, HL68525)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Tuberculosis Research and Epidemiology
  • Life Sciences › Immunology and Microbiology › Immunology › Immune responses and vaccinations
  • Health Sciences › Medicine › Infectious Diseases › Infectious Diseases and Tuberculosis
Keywords
Mycobacterium tuberculosis, IDO1, tryptophan metabolism, tryptophan catabolism, picolinic acid, quinolinic acid, macrophages, dendritic cells, tuberculosis immunology, bacterial growth inhibition
MeSH terms
Animals, Female, Humans, Immunohistochemistry, Lung, Macrophages, Mice, Inbred C57BL, Mycobacterium tuberculosis, Picolinic Acids, T-Lymphocytes, Tryptophan, Tuberculosis, Blotting, Western, DNA Primers, Mice, Knockout, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling, Microarray Analysis, Indoleamine-Pyrrole 2,3,-Dioxygenase, Mice, Kaplan-Meier Estimate, Real-Time Polymerase Chain Reaction

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