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Nature Immunology | Article

Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells

Journal name:
Nature Immunology
Volume:
12,
Pages:
870–878
Year published:
DOI:
doi:10.1038/ni.2077
Received
Accepted
Published online

Abstract

Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-γ is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-β (TGF-β) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-β-driven tolerance in noninflammatory contexts.

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  1. IDO catalytic activity is not required for the IDO-dependent, immunoregulatory effects of pDCs conditioned with TGF-[beta] in vitro.
    Figure 1: IDO catalytic activity is not required for the IDO-dependent, immunoregulatory effects of pDCs conditioned with TGF-β in vitro.

    (a) Proliferation of CD4+ T cells in a CD4+CD25− T cell population cultured for 4 d together with pDCs transfected with scrambled sequence (Control) or siRNA targeting Ido1 (two separate siRNAs: Ido1-1 and Ido1-2) or Ido2, or treated with 1-MT; cultures were left untreated (None) or treated with IFN-γ or TGF-β and were assessed by cytofluorometry as the frequency of cells positive for the thymidine analog EdU. (b) Apoptosis of CD4+ T cells in 24-hour cocultures established as in a, assessed as the frequency of cells positive for propidium iodide (PI) and annexin V (AnnV). (c) Expression of Foxp3 in 4-day cocultures as in a, assessed by cytofluorometry as the frequency of CD4+Foxp3+ cells. Histograms and plots for a–c are in Supplementary Figures 2–4. (d) Proliferation of CD4+CD25− T cells cultured as in a in various numbers (horizontal axis), in the presence of soluble anti-CD3 and irradiated populations of splenocytes depleted of T cells. (e) Cytokines in supernatants of 4-day cocultures as in a. NC, siRNA with a scrambled sequence (negative control). (f) TGF-β1 expression on pDCs from wild-type C57BL/6 mice (WT) or Ido1−/− mice after 24 h of treatment with TGF-β or medium alone (Control). Numbers in top right quadrants indicate percent TGF-β+ cells positive for the antibody 120G8 (to pDCs). Compiled results (mean ± s.d.): TGF-β, 4.5 ± 1.5; wild-type control, 1.2 ± 0.4 (P = 0.011 (Student's t-test)). *P < 0.01 and **P < 0.001 (Student's t-test). Data are from three independent experiments (mean and s.d. in a–c,e) or are from one experiment representative of three (d,f; mean and s.d. of triplicates in d).

  2. The catalytic activity of IDO is not required for IDO-dependent, immunoregulatory effects induced in vivo by pDCs conditioned with TGF-[beta].
    Figure 2: The catalytic activity of IDO is not required for IDO-dependent, immunoregulatory effects induced in vivo by pDCs conditioned with TGF-β.

    (a) In vivo suppression of the activity of HY-pulsed C57BL/6 CD8− DCs transferred into recipient mice coexpressing enhanced green fluorescent protein and Foxp3, either alone (None) or in combination with a minority fraction (5%) of pDCs with no conditioning (pDCs) or conditioned with IFN-γ (pDCs + IFN-γ) or TGF-β (pDCs + TGF-β) that were left untransfected (Control), transfected with siRNA with a scrambled sequence (negative control (NC)) or targeting Ido1 or Ido2, or treated with 1-MT, or treated with anti-TGF-β at the time of DC sensitization; analysis of skin reactivity of recipient mice to the eliciting peptide at 15 d is presented as change in footpad weight. (b) Histology of sections of footpads from mice sensitized for 15 d with pDCs (left untreated (No treatment) or treated with IFN-γ or TGF-β), then injected in the footpad with peptide (Experimental footpad) or vehicle alone (Control footpad); footpads removed 24 h later were stained with hematoxylin and eosin for analysis of leukocyte infiltration. Bottom, photographs of footpads. Scale bars, 100 μm. (c) Production of IFN-γ by leukocytes from mice (n = 6 per group) sensitized with peptide-pulsed DCs (left untreated (None) or treated with IFN-γ or TGF-β), treated as in a (key), and challenged with HY 15 d later in the footpad; leukocytes collected 24 h later from popliteal lymph nodes (draining left hind footpads) were restimulated for 48 h in vitro with HY, followed by analysis of IFN-γ in culture supernatants. (d) Expression of Foxp3 by cells from popliteal lymph nodes obtained as in c, assessed by cytofluorometry as the frequency of CD4+Foxp3+ cells. *P < 0.01 (Student's t-test). Data are representative of four (a,d), two (b) or three (c) experiments (mean and s.d. in a,c,d).

  3. TGF-[beta] induces the formation of IDO-SHP-1-SHP-2 complexes and activation of SHP-1 phosphatase activity in pDCs.
    Figure 3: TGF-β induces the formation of IDO–SHP-1–SHP-2 complexes and activation of SHP-1 phosphatase activity in pDCs.

    (a) Kinetics of the phosphorylation of IDO ITIM2 in pDCs conditioned with TGF-β or IFN-γ, analyzed by immunoblot sequentially with antibody to phosphorylated ITIM2 (p-IDO), anti-IDO and anti-β-tubulin. (b) Real-time PCR analysis of Ptpn6, Ptpn11 and Inpp5d transcripts in pDCs treated as in a, normalized to the expression of Gapdh (encoding glyceraldehyde phosphate dehydrogenase) and presented relative to results in untreated cells (dotted line, onefold). *P < 0.01 (Student's t-test). (c) Precipitation (Ppt) of SHP-1, SHP-2 and SHIP from lysates of untreated P1 cells and TGF-β-treated pDCs with unphosphorylated IDO peptides (ITIM1 and ITIM2) or tyrosine-phosphorylated IDO peptides (p-ITIM1 and p-ITIM2), analyzed by immunoblot sequentially with anti-SHP-1, anti-SHP-2 and anti-SHIP. WCL (below), immunoblot analysis of whole-cell lysates as above. (d) Immunoprecipitation (IP) of proteins from lysates from pDCs (left untreated (−) or treated for 24 h with TGF-β) with anti-IDO, analyzed by immunoblot sequentially with anti-SHP-1, anti-SHP-2 and anti-IDO. WCL (right), immunoblot analysis of whole-cell lysates (one-tenth of the full volume), as a loading control. (e) Phosphatase activity in anti-IDO immunoprecipitates of pDCs treated as in d alone (−) or also transfected with negative control siRNA (NC) or siRNA targeting Ido1, Ptpn6 or Ptpn11 or treated with stibogluconate (Stibo); results are presented as free phosphate released from samples with a volume of 50 μl. *P < 0.01 and **P < 0.001 (Student's t-test). (f) Phosphatase activity (top) and kynurenine production (bottom) by P1 cells left untransfected (−), mock-transfected (Control), or transfected with constructs for wild-type IDO (IDO), IDO2, or IDO mutants lacking the histidine residue required for catalytic activity (IDO(H350A)) or the ITIM1 tyrosine (IDO(Y115F)), the ITIM2 tyrosine (IDO(Y253F)) or both (Y115F,Y253F)), then left untreated (open bars) or treated (filled bars) with stibogluconate (top) or 4 μM 1-MT (bottom). *P < 0.05 and **P < 0.005 (top) or **P < 0.001 (bottom; all Student's t-test). ND, not detectable. (g) Intracellular immunofluorescence analysis of the colocalization of IDO and SHP-1 in P1 cells transfected as in f, treated for 10 min with Na3VO4 and fixed with formaldehyde; Alexa Fluor 488–labeled anti-IDO was used in combination with anti-SHP-1 and indocarbocyanine-conjugated antibody to mouse immunoglobulin, and nuclei were stained with the DNA-intercalating dye DAPI (blue). Data are from one experiment representative of three (a,c,d,f,g) or are from three experiments (b,e; mean and s.d. in b,e,f).

  4. IDO phosphorylation requires PI(3)K-dependent but Smad-independent TGF-[beta] signaling events and is mediated by Fyn but not Syk.
    Figure 4: IDO phosphorylation requires PI(3)K-dependent but Smad-independent TGF-β signaling events and is mediated by Fyn but not Syk.

    (a) Phosphorylation of IDO ITIM2 in pDCs pretreated for 1 h with SIS3 or LY294002 before incubation for 60 min with TGF-β, assessed by sequential immunoblot analysis with antibody to phosphorylated ITIM2 (p-IDO), anti-IDO and anti-β-tubulin. (b) Real-time PCR analysis of Ptpn6 transcripts in pDCs treated for 16 h with TGF-β with or without SIS3 or LY294002, normalized (as in Fig. 3b) and presented relative to results in untreated cells. *P < 0.001 (Student's t-test). (c) Distribution of absolute expression of genes encoding mouse Src kinases (horizontal axis) in untreated mouse pDCs (n = 17), based on data derived from publicly available gene expression data sets. Each symbol represents an individual pDC gene; small horizontal lines indicate the mean. (d) Heat map of standardized expression of genes as in c (all values relative to an arbitrary unit scale) in untreated mouse pDCs (n = 17) derived from six different sets of samples from the Gene Expression Omnibus (numbers above lanes, accession codes for the 17 samples). (e) Immunoblot analysis of Fyn in lysates of pDCs left untreated (−) or treated for 24 h with TGF-β (+). (f) Phosphorylation of IDO ITIM2 (as in a) in pDCs treated for 1 h with PP2, PP3 or a Syk inhibitor before incubation for 60 min with TGF-β. Data are from one experiment representative of two (a,e,f) or three (b; error bars, s.d.) or are from one metanalysis (c,d).

  5. IDO and SHP proteins drive a signaling pathway in pDCs that involves activation of the noncanonical NF-[kappa]B pathway and production of type I interferon.
    Figure 5: IDO and SHP proteins drive a signaling pathway in pDCs that involves activation of the noncanonical NF-κB pathway and production of type I interferon.

    (a) Activation of IKKα and IKKβ in pDCs treated for 0–180 min (above lanes) with TGF-β or IFN-γ; lysates were sequentially blotted with antibody to phosphorylated IKKα or IKKβ, anti-IKKα and anti-IKKβ (labels between blots indicate expected migration). (b) Enzyme-linked immunosorbent assay of the activation of p65, p52 and RelB in nuclear extracts of pDCs not treated with cytokine (None) or treated with cytokine for 30 or 60 min as in a (bottom), and not transfected or pretreated (None) or pretreated for 1 h with 1-MT or transfected with negative control siRNA or siRNA targeting Ido1, Ido2, Ptpn6 and/or Ptpn11 (horizontal axis); results are presented as absorbance at 450 nm (A450). *P < 0.05 and **P < 0.01, cytokine-treated versus untreated (Student's t-test). (c) Enzyme-linked immunosorbent assay of IFN-α in supernatants of BALB/c, C57BL/6 or Ido1−/− pDCs left untreated or treated for 24 h with various combinations of IFN-γ or TGF-β and 1-MT (key), plus no siRNA (None), negative control siRNA or siRNA targeting various genes (horizontal axis; two separate siRNAs for Ido1 siRNA (as in Fig. 1a) and two for Irak1). *P < 0.01 and **P < 0.001 (Student's t-test). (d) Coimmunoprecipitation of IRAK1 and IDO with SHP proteins (with a mixture of anti-SHP-1 and anti-SHP-2) from lysates of pDCs transfected with the Ido1-containing construct, left untreated or treated for 24 h with TGF-β, followed by sequential immunoblot analysis with anti-IDO, anti-IRAK1, anti-SHP-1 and anti-SHP-2. Data are from one experiment representative of two (d) or three (a) or are from three experiments (b,c; mean and s.d.).

  6. IDO-dependent immunoregulatory effects of pDCs conditioned with TGF-[beta] are mediated by Fyn, SHP proteins, the noncanonical NF-[kappa]B pathway and type I interferon signaling.
    Figure 6: IDO-dependent immunoregulatory effects of pDCs conditioned with TGF-β are mediated by Fyn, SHP proteins, the noncanonical NF-κB pathway and type I interferon signaling.

    (a) Frequency of CD4+Foxp3+ cells in a CD4+CD25− T cell population cultured for 4 d together with BALB/c wild-type or 129/Sv wild-type or Ifnar−/− pDCs transfected with siRNA (horizontal axis) and treated with TGF-β. *P < 0.01 (Student's t-test). (b) IL-6 in supernatants of cocultures as in a. (c) Real-time PCR analysis of Tgfb1 transcripts in pDCs transfected with siRNA (horizontal axis) and treated for 16 h with TGF-β; normalized results (as in Fig. 3b) are presented relative to results in untreated cells (dashed line, onefold). (d) Skin-test assay (as in Fig. 2a) of the in vivo suppressive activity of TGF-β-treated BALB/c pDCs pretreated for 1 h with PP2 or PP3 (key) or transfected with siRNA (horizontal axis), and TGF-β-treated untransfected 129/Sv wild-type or Ifnar−/− pDCs loaded with IGRP. *P < 0.001 (Student's t-test). Data are from three experiments (mean and s.d.).

  7. TGF-[beta] induces long-term expression of IDO in pDCs, but IFN-[gamma] does not.
    Figure 7: TGF-β induces long-term expression of IDO in pDCs, but IFN-γ does not.

    (a) Chromatin-immunoprecipitation assay of the binding of p52-RelB, IRF3, IRF4 (negative control), IRF7 and IRF8 to the mouse Ido1 promoter in pDCs treated for 3 h (p52-RelB) or 16 h (IRFs) with TGF-β, quantified by real-time PCR with primers composed of the putative noncanonical (NC) NF-κB-, ISRE-1- or ISRE-2-binding region; normalized results (as in Fig. 3b) are presented relative to results in untreated cells (dashed line, onefold). (b) Time course of activation of the Ido1 promoter in pDCs transfected with a firefly luciferase construct of the Ido1 promoter plus noncoding sequence in Ido1 exon 1 and incubated for 3–48 h with IFN-γ or TGF-β; results are normalized to the activity of a cotransfected constitutive reporter and are presented relative to those in untreated cells (dashed line, onefold). (c) Ido1 promoter activity in wild-type BALB/c, wild-type (129/Sv) or Ifnar−/− 129/Sv, wild-type (C57BL/6) or Ido1−/− pDCs left untransfected (None) or transfected with negative control siRNA or gene-specific siRNA (horizontal axis), then incubated for 24 h with TGF-β; luciferase activity was assessed as in b. (d) Immunoblot analysis of the time course of IDO protein expression in pDCs either incubated for 0, 24 or 48 h (above lanes) with TGF-β or IFN-γ, or incubated for 24 h with TGF-β or IFN-γ, washed extensively and incubated for 24 h with medium alone (w); lysates were probed sequentially with anti-IDO and anti-β-tubulin. (e) Time-course of IDO catalytic activity in pDCs left untreated (None; time 0) or treated for 16, 24 or 48 h with IFN-γ or TGF-β for 16–48 h, or treated with washing as in d (Wash), assessed as kynurenine in culture supernatants. *P < 0.01 (Student's t-test). Data are from three experiments (a–c,e; mean and s.d.) or are from one experiment representative of three (d).

  8. Long-term immunoregulatory effects in vivo of pDCs conditioned by TGF-[beta] but not those conditioned by IFN-[gamma].
    Figure 8: Long-term immunoregulatory effects in vivo of pDCs conditioned by TGF-β but not those conditioned by IFN-γ.

    (a) Long-term skin-test assay of mice coexpressing enhanced green fluorescent protein and Foxp3 under the control of an endogenous promoter (B6.Cg-Foxp3 mice; given C57BL/6 pDCs) or C57BL/6 mice (given Ido1−/− pDCs) given transfer of HY-pulsed pDCs left untreated (None) or pretreated for 24 h with IFN-γ or TGF-β (C57BL/6 or Ido1−/−) and combined with HY-pulsed CD8− DCs, assessed by reactivity to a skin test 3 months later. *P < 0.001 (Student's t-test). (b) Cytofluorometry of the frequency of CD4+Foxp3+ cells among leukocytes from the popliteal lymph nodes (draining the left hind footpads) of mice (n = 6 per group) sensitized as in a with peptide-pulsed DCs 3 months earlier and challenged in the footpad with peptide 24 h before analysis. (c) IFN-γ in leukocytes from popliteal lymph nodes collected as in b, assessed after restimulation for 48 h in vitro with HY. (d) Cytofluorometry of the frequency of CD4+Foxp3+ cells (numbers in top right quadrants) among CD4+CD25− T cells cultured for 4 d together with TGF-β-treated pDCs, plus no antibody (No Ab; left)), anti-TGF-β (middle) or isotype-matched control antibody (isotype; right) added at the beginning of culture (time 0) or at 48 h (right margin). *P < 0.01 and (Student's t-test). Data are from three experiments (a–c; mean and s.d.) or are from one experiment representative of two (d).

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Author information

  1. These authors contributed equally to this work.

    • Francesca Fallarino,
    • Paolo Puccetti &
    • Ursula Grohmann

Affiliations

  1. Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy.

    • Maria T Pallotta,
    • Ciriana Orabona,
    • Claudia Volpi,
    • Carmine Vacca,
    • Maria L Belladonna,
    • Roberta Bianchi,
    • Mario Calvitti,
    • Maria C Fioretti,
    • Francesca Fallarino,
    • Paolo Puccetti &
    • Ursula Grohmann

  2. Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy.

    • Giuseppe Servillo &
    • Cinzia Brunacci

  3. Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy.

    • Silvio Bicciato &
    • Emilia M C Mazza

  4. Bioceros, Utrecht, The Netherlands.

    • Louis Boon

  5. Institute for Research in Biomedicine, Bellinzona, Switzerland.

    • Fabio Grassi

Contributions

M.T.P. designed and did experiments; C.O., C.Vo. C.Va., M.L.B., R.B., C.B., M.C. and E.M.C.M. did experiments; G.S., S.B. and M.C.F. contributed to experimental design; L.B. and F.G. provided reagents; F.F. designed experiments and supervised research; P.P. supervised research; and U.G. designed experiments, supervised research and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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