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. 2008 May 6;105(18):6690-5.
doi: 10.1073/pnas.0708809105. Epub 2008 Apr 24.

Indoleamine 2,3-dioxygenase in lung dendritic cells promotes Th2 responses and allergic inflammation

Hui Xu  1 , Timothy B Oriss, Mingjian Fei, Adam C Henry, Barbro N Melgert, Li Chen, Andrew L Mellor, David H Munn, Charles G Irvin, Prabir Ray, Anuradha Ray
Affiliations

Indoleamine 2,3-dioxygenase in lung dendritic cells promotes Th2 responses and allergic inflammation

Hui Xu et al. Proc Natl Acad Sci U S A. .

Abstract

Indoleamine 2,3 dioxygenase (IDO) has emerged as an important mediator of immune tolerance via inhibition of Th1 responses. However, the role of IDO in antigen-induced tolerance or allergic inflammation in the airways that is regulated by Th2 responses has not been elucidated. By using IDO(-/-) mice, we found no impairment of airway tolerance, but, surprisingly, absence of IDO provided significant relief from establishment of allergic airways disease, as evident from attenuated Th2 cytokine production, airway inflammation, mucus secretion, airway hyperresponsiveness, and serum ovalbumin-specific IgE. Myeloid dendritic cells isolated from lung-draining lymph nodes of mice immunized for either Th1 or Th2 response revealed fewer mature dendritic cells in the lymph nodes of IDO(-/-) mice. However, the net functional impact of IDO deficiency on antigen-induced responses was more remarkable in the Th2 model than in the Th1 model. Collectively, these data suggest that IDO is not required for the induction of immune tolerance in the airways but plays a role in promoting Th2-mediated allergic airway inflammation via unique effects on lung dendritic cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
IDO−/− mice show attenuated Th2-type airways inflammation in chronic experimental asthma. Animals were subjected to a model of chronic airway inflammation consisting of periodic exposure to OVA over an extended period. (A) Total (Left) and differential (Right) cell counts. Macs, macrophages; Lymphs, lymph nodes; PMNs, polymorphonuclear leukocytes; Eos, eosinophils. (B) OVA-specific serum IgE as determined by ELISA. Data shown are mean ± SD. (C) Cytokine in lung homogenates as determined by multiplex assay. Data shown are mean ± SD. (D) AHR in response to inhaled MCh challenge (n = 6 mice per group). Both WT and IDO−/− mice showed increased AHR in OVA-challenged mice in response to inhaled MCh in all indices of lung function as compared with naive MCh-challenged mice. Rn, G, and H were all lower in OVA-challenged IDO−/− mice with respect to similarly challenged WT mice, and the difference reached statistical significance as indicated. (E) Production and secretion of mucus in lung tissue by periodic acid-Schiff reagent staining. Arrows denote inflammation (green) and mucus production (red). Magnification, ×100. Assessment of mucus plugging of the airways of mice from the two groups was based on the evaluation of both proximal and distal airways (n = 6–7 mice per group). Values are mean ± SD. ∗, P < 0.05. Data shown are representative of two independent experiments.
Fig. 2.
Fig. 2.
Reduced T cell expansion in lung-draining lymph nodes and reduced Th2 cytokine production in lung tissue in IDO−/− mice. (A) IDO−/− and WT animals received OVA/CT for three consecutive days via the intranasal route. Five days after the last treatment, lung-draining LN cells were isolated. Total LN cell counts representative of five independent experiments are shown. The lines connect age-matched animals for each individual experiment. The horizontal bars indicate the mean total cell count for the five experiments. ∗, P < 0.05. (B) OVA-specific serum IgE was determined by ELISA. Data shown are mean ± SD. ∗, P < 0.05. (C) Total LN cells were incubated with OVA, and proliferation was assessed by [3H]thymidine incorporation. Data shown are representative of two independent experiments. (D) DO11.10 TCR transgenic CD4+ T cells were labeled with CFSE and adoptively transferred intravenously into naive wild-type or IDO−/− animals. The mice received OVA/CT for two consecutive days. CFSE dilution in transferred T cells expressing the clonotypic TCR, as recognized by KJ1–26 mAb, was evaluated 1 day later by flow cytometry. The data show the equivalent percentage of proliferation in each generation of cells derived from WT and IDO−/− mice. However, the total count of CFSE-labeled cells, as shown in the right, was lower in the lymph nodes of IDO-deficient mice. (E) Cytokine levels in lung tissue of the same animals at the early time point after transfer. All data shown are mean ± SD. ∗, P < 0.05. Data shown are representative of three independent experiments. (F) Inhibition of allergic response upon treatment with 1-MT. Pellets containing 1-MT (releasing 10 mg/day) or placebo were implanted under the dorsal skin in BALB/c mice 1 day before sensitization. OVA/CT was instilled on three successive days as described in (A). On day 5 after the last instillation, the animals were challenged by aerosolized 1% OVA for seven consecutive days. 24 h after the last aerosol treatment, lungs were lavaged, and blood was collected. The results shown are mean cell number per ml of bronchoalveolar lavage (BAL) fluid per mouse ± SD. OVA-specific IgE and IgG2a levels in the sera were measured by ELISA. Data shown are means of Ig levels in four mice per group ± SD and are representative of two independent experiments. Macs, macrophages; Lymphs, lymph nodes; PMNs, polymorphonuclear leukocytes; Eos, eosinophils.
Fig. 3.
Fig. 3.
Fewer mature mDCs in lung-draining lymph nodes of immunized IDO−/− mice and a corresponding decrease in the ability to stimulate Th2 cytokine production. (A) IDO expression in purified lung DCs from WT and IDO−/− mice as determined by immunoblotting. Expression of β-actin was monitored as a loading control. (B) mDCs from lung-draining LNs were partially purified before analysis by flow cytometry. The mDCs were identified by expression of CD11c and high-level MHC class II expression, as described previously (25), and were then assessed for expression of the indicated costimulatory molecules. (C) Based on expression of CD11c and MHC class II, lung-draining LN mDCs were purified by cell sorting 1 day after the last of three daily administrations of OVA/CT. The sorted mDCs were cultured with CD4+CD25− T cells from naive DO11.10 TCR transgenic mice in the presence of specific OVA peptide for 5 days, at which time cytokines secreted in the culture supernatant were assessed by multiplex assay. (D) mDCs from lung-draining LNs were similarly purified 5 days after the last of three daily administrations of OVA/CT and were cocultured with CD4+ T cells purified from the same LN with whole OVA protein (100 μg/ml). Cytokines secreted in the culture supernatants after 4 days were assessed by multiplex assay. ∗, P < 0.05. The experiment was repeated, yielding similar results.
Fig. 4.
Fig. 4.
IDO deficiency impacts antigen-induced Th2 responses more than Th1 responses. Wild-type and IDO −/− mice received three daily intranasal administrations of OVA/CT or OVA/CpG. (A) Lungs and lung-draining lymph nodes were harvested from four or five animals per group 5 days after the last intranasal treatment. (B and C) The remaining animals were rested for 3 days, given an intranasal boost with OVA/CT or CpG, rested an additional 2 days, and then challenged with aerosolized OVA for ten consecutive days. (A) Immediately after isolation, the LN and lung T cells were subjected to ELISPOT assay after brief stimulation with phorbol 12-myristate 13-acetate (25 ng/ml) and ionomycin (500 ng/ml). The results are expressed as the number of cytokine-expressing cells per mouse based on the number of CD4+ T cells recovered (see Fig. S4). (B) One day after the last challenge with aerosolized OVA, BAL was performed to determine the total number of cells in the BAL fluid (Left), and differential cell counts were performed (Center and Right). Macs, macrophages; Lymphs, lymph nodes; PMNs, polymorphonuclear leukocytes; Eos, eosinophils. (C) OVA-specific serum IgE and IgG2a concentrations were determined by ELISA by using sera from blood collected 1 day after the immunization plus challenge protocol. Data shown are means of Ig levels in four mice per group ± SD.∗, P < 0.05. Shown is an experiment representative of two.
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