Airway epithelial indoleamine 2,3-dioxygenase inhibits CD4+ T cells during Aspergillus fumigatus antigen exposure

Abstract

Indoleamine 2,3-dioxygenase (IDO) suppresses the functions of CD4(+) T cells through its ability to metabolize the essential amino acid tryptophan. Although the activity of IDO is required for the immunosuppression of allergic airway disease by the Toll-Like-Receptor 9 (TLR9) agonist, oligonucleotides comprised of cytosine and guanine nucleotides linked by phosphodiester bonds (CpG) DNA, it is unclear whether IDO expression by resident lung epithelial cells is sufficient to elicit these effects. Therefore, we created a transgenic mouse inducibly overexpressing IDO within nonciliated airway epithelial cells. Upon inhalation of formalin-fixed Aspergillus fumigatus hyphal antigens, the overexpression of IDO from airway epithelial cells of these mice reduced the number of CD4(+) T cells within the inflamed lung and impaired the capacity of antigen-specific splenic CD4(+) effector T cells to secrete the cytokines IL-4, IL-5, IL-13, and IFN-γ. Despite these effects, allergic airway disease pathology was largely unaffected in mice expressing IDO in airway epithelium. In support of the concept that dendritic cells are the major cell type contributing to the IDO-inducing effects of CpG DNA, mice expressing TLR9 only in the airway epithelium did not augment IDO expression subsequent to the administration of CpG DNA. Furthermore, the systemic depletion of CD11c(+) cells rendered mice incapable of CpG DNA-induced IDO expression. Our results demonstrate that an overexpression of IDO within the airway epithelium represents a novel mechanism by which the number of CD4(+) T cells recruited to the lung and their capacity to produce cytokines can be diminished in a model of allergic airway disease, and these results also highlight the critical role of dendritic cells in the antiasthmatic effects of IDO induction by CpG DNA.

Figure 1.

Kynurenine production and CD4⁺ T-cell proliferation attributable to indolamine 2,3-dioxygenase (IDO) expression from transformed mouse airway epithelial cells. Transformed mouse airway epithelial cells (MTCCs) were transduced with a control adenovirus expressing LacZ (AdLacZ) or increasing amounts (VP, viral particles) of adenovirus expressing IDO (AdIDO). (A) We used 600 μM 1-methyl tryptophan (1-MT) to inhibit IDO activity. An IDO activity assay was performed to measure the production of kynurenine. MTCCs transduced with AdIDO, a control adenovirus (AdLacZ), or left untransduced (No Virus) were cocultured with CD4⁺ T cells isolated from spleens of wild-type mice and stimulated in vitro with anti-CD3 and anti-CD28. (B) CD4⁺ T-cell proliferation was measured by tritiated thymidine incorporation. Values are means (± SEM) of three wells/condition, and are representative studies performed twice.

Figure 2.

CC10-IDO mouse characterization. CC10-IDO triple-transgenic mice were created by the genomic incorporation of three transgenes. (A) One transgene encodes an activator of transcription, whereby transcription is directed by the rat CC10 (rCC10) promoter, restricting transcription to nonciliated airway epithelial cells. The second transgene encodes a suppressor of transcription also directed by the rat CC10 promoter. The third transgene encodes hemagglutinin (HA)-tagged IDO directed by a doxycycline-sensitive tetracycline promoter. TetOP, tetracycline-regulated operator. (B) After 14 days of doxycycline feeding, RNA was isolated from whole lungs of CC10-IDO transgenic (Tg⁺) and transgene-negative (Tg⁻) littermates, and quantitative RT-PCR was performed to measure IDO induction from various tissues. (C) IDO mRNA induction was measured over time from Tg⁺ and Tg⁻ littermates after consuming doxycycline food for up to 28 days. (D) HPLC was performed to measure IDO activity from whole-lung homogenate of Tg⁺ and Tg⁻ mice after 28 days of consuming doxycycline food. (E) Representative lung sections from doxycycline-fed CC10-IDO Tg⁺ (left) and Tg⁻ (right) littermates were analyzed for HA (orange) staining in airway epithelium. Values are means (± SEM) of 5 mice/group.

Figure 3.

Pulmonary gene expression, serum IgE, and airway physiology after Aspergillus fumigatus antigen sensitization and challenge. CC10-IDO transgenic (Tg⁺) and transgene-negative (Tg⁻) mice aspirated 1 μg of formalin-fixed A. fumigatus hyphal extract on days 0, 7, and 14. Mice were analyzed on day 19. All mice consumed doxycycline-containing food starting on day 0 and throughout the entire protocol. RNA from whole lung was analyzed by quantitative RT-PCR, and IDO (A) and Muc5AC (B) expression were normalized to glyceraldehyle 3-phosphade dehydrogenase (GAPDH), relative to that measured from the lungs of Tg⁻ mice. (C) Serum was analyzed by ELISA to quantitate total IgE concentrations. Tg⁺ (solid triangles) and Tg⁻ (open squares) mice sensitized to A. fumigatus antigens were assessed on day 19 for airway hyperresponsiveness to increasing amounts of inhaled methacholine and compared with naive BALB/cJ mice (solid circles). Measurements were recorded from forced oscillations and determined as airway resistance (R_N), tissue damping (G), and tissue stiffness (H). (D) As derived from the constant phase model, data are expressed as the percent of baseline measurements (± SEM) for each of the parameters measured from 9 Tg⁺, 8 Tg⁻, and 7 naive BALB/cJ mice, and are representative of two independent studies.

Figure 4.

BAL cell differential count after Aspergillus fumigatus antigen sensitization and challenge. After sensitization and challenge with 1 μg of formalin-fixed A. fumigatus hyphal extract, saline was instilled into the lungs of tracheotomized, naive, CC10-IDO transgenic (Tg⁺) and transgene-negative (Tg⁻) mice that subsequently recovered, and total (A) and differential (B) cell counts were performed. All mice consumed doxycycline-containing food throughout the entire protocol for 19 days. Values are means (± SEM) of 10 Tg⁺ mice and 8 Tg⁻ mice, and are representative studies performed twice.

Figure 5.

Lymphocyte populations within the lung after Aspergillus fumigatus antigen sensitization and challenge. Lung digests were performed from CC10-IDO transgenic mice (Tg⁺) (bottom histograms) and transgene-negative littermates (Tg⁻) (top histograms) that had been sensitized and challenged with A. fumigatus antigens. Flow cytometry was performed to determine the frequency of B cells, T cells, CD4⁺ T cells, CD8⁺ T cells, and natural killer T (NKT) lymphocytes found within the lymphocyte population. Below the representative histograms (A), graphs display the frequency of cells as determined from the gated lymphocyte parent population (B), and cell numbers were calculated from the frequency multiplied by the lymphocyte numbers in the lungs (C). Data from 10 Tg⁺ and 8 Tg⁻ mice/group (± SEM) are presented in the graphs below the representative histograms.

Figure 6.

Aspergillus-specific CD4⁺ T-cell cytokine secretion. Positively selected splenic CD4⁺ T cells from CC10-IDO transgenic (Tg⁺) and transgene-negative (Tg−) littermate mice were cultured in the presence of antigen-presenting cells isolated from naive BALB/cJ mice and 1 μg of Aspergillus crude hyphal extract for 72 hours. Cytokine concentrations were measured by ELISA. Values are means (± SEM) of 8 Tg⁺ mice and 8 Tg⁻ mice, and are representative of experiments performed twice.

Figure 7.

Lung IDO expression in CC10-hTLR9 mice exposed to CpG DNA. (A) Tissues from CC10-hTLR9 transgenic mice on a TLR9-deficient background were analyzed by RT-PCR for lung-specific human Toll-Like-Receptor 9 (hTLR9) transgene expression. Transgene-negative (Tg⁻) and CC10-hTLR9 transgenic (CC10-hTLR9 Tg⁺) mice were administered 10 μg CpG DNA into their airways by oropharyngeal aspiration. Twenty-four hours later, pulverized lungs were analyzed by quantitative RT-PCR for relative expression levels of hTLR9 transgene (B), mouse TLR9 (mTLR9) (C), CCL20 (D), and IDO (E). Values are means (± SEM) of five mice per group, and are representative of experiments performed twice.

Figure 8.

Lung IDO expression in mice depleted of conventional myeloid dendritic cells and exposed to CpG DNA. Transgene-negative (Tg⁻) and CD11c-DTR transgenic mice (Tg⁺) were administered 4 ng/g diphtheria toxin via oropharyngeal aspiration, to deplete dendritic cells locally from the lung. Twenty-four hours later, mice were administered 50 μg non-CpG DNA or CpG DNA into their airways by intraperitoneal injection. (A) Lung homogenates were analyzed 24 hours later by Western blot for IDO expression. (B) IDO band intensities on Western blots were quantitated by luminescence. (C) Pulverized lungs were analyzed by quantitative RT-PCR for relative expression levels of IDO. Values are means (± SEM) of three mice per group, and are representative of experiments performed twice.