A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity

Abstract

Airway hypersensitive reaction (AHR) is an animal model for asthma, which is caused or enhanced by environmental factors such as allergen exposure. However, the precise mechanisms that drive AHR remain unclear. We identified a novel subset of natural killer T (NKT) cells that expresses the interleukin 17 receptor B (IL-17RB) for IL-25 (also known as IL-17E) and is essential for the induction of AHR. IL-17RB is preferentially expressed on a fraction of CD4(+) NKT cells but not on other splenic leukocyte populations tested. IL-17RB(+) CD4(+) NKT cells produce predominantly IL-13 and Th2 chemokines upon stimulation with IL-25 in vitro. IL-17RB(+) NKT cells were detected in the lung, and depletion of IL-17RB(+) NKT cells by IL-17RB-specific monoclonal antibodies or NKT cell-deficient Jalpha18(-/-) mice failed to develop IL-25-dependent AHR. Cell transfer of IL-17RB(+) but not IL-17RB(-) NKT cells into Jalpha18(-/-) mice also successfully reconstituted AHR induction. These results strongly suggest that IL-17RB(+) CD4(+) NKT cells play a crucial role in the pathogenesis of asthma.

Figure 1.

Phenotypic and gene expression profiles of splenic IL-17RB⁺ NKT cells. (A) Identification of IL-17RB⁺ cells from BALB/c mice. The indicated populations were gated and analyzed by FACS using F(ab′)₂ fragments of the anti–IL-17RB mAb B5F6. Shaded profiles in the histograms indicate the background staining with rat F(ab′)₂ IgG2a. (B) FACS profile of CD1d dimer⁺ NKT cells stained with anti-CD4 and anti–IL-17RB. (C) Surface phenotype of DN, IL-17RB⁻ CD4⁺, and IL-17RB⁺ CD4⁺ NKT cells indicated in B. Shaded profiles in the histograms indicate the background staining with isotype-matched control antibody. Percentages are shown. (D–H) Quantitative analyses of genes for chemokine receptors (D), Th1-related molecules (E), Th2-related molecules (F), cytotoxic molecules (G), and Th17-related molecules (H) in isolated DN (black bars), IL-17RB⁻ CD4⁺ (gray bars), and IL-17RB⁺ CD4⁺ (white bars) NKT cells. Expression of each mRNA was determined by quantitative real-time PCR using the primer sets shown in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20080698/DC1) and is depicted as the number of transcripts per one copy of the housekeeping gene HPRT. One representative out of three experiments is shown (means ± SEM).

Figure 2.

Properties of splenic IL-17RB⁺ CD4⁺ NKT cells. (A and B) Proliferation (A) and cytokine production (B) of DN, IL-17RB⁻ CD4⁺, and IL-17RB⁺ CD4⁺ NKT cells. Indicated NKT cell subpopulations (10⁶ cells/ml) were co-cultured for 3 d with or without splenic CD11c⁺ DCs (10⁵ cells/ml) in the absence or presence of IL-25 (0, 1, and 10 ng/ml), and proliferation was assayed by [³H]thymidine incorporation (A) or cytokine production by cytokine bead array (B). The data are shown as the means ± SD of three cultures. The data are representative of four independent experiments. (C) Quantitative analysis of genes in IL-17RB⁺ CD4⁺ NKT cells. IL-17RB⁺ CD4⁺ NKT cells (10⁶ cells/ml) were co-cultured with splenic CD11c⁺ DCs (10⁵ cells/ml) in the presence of 10 ng/ml IL-25 for 48 h. IL-17RB⁺ CD4⁺ NKT cells were sorted and analyzed for their gene expression by quantitative real-time PCR, as described in Materials and methods. The data are representative of three independent experiments (means ± SEM).

Figure 3.

Involvement of IL-17RB⁺ NKT cells in the development of IL-25–induced AHR. (A and B) Tissue distribution of IL-17RB⁺ NKT cells (A) and IL-17RB expression among lung mononuclear cell populations (B) in BALB/c mice. α-GalCer/CD1d dimer⁺ NKT cells in the indicated organs (A) and mononuclear cell populations in the lung (B) from BALB/c mice were gated and analyzed by FACS using B5F6 F(ab′)₂ IL-17RB mAb. Shaded profiles in the histograms indicate the background staining with rat F(ab′)₂ IgG2a (n = 3). Percentages are shown. (C) Development of AHR. The changes in R_L were measured. The detailed method for development of OVA/IL-25–induced AHR is described in Materials and methods. Results are expressed as the mean ± SEM. The group of IL-25–treated WT mice was compared with three other groups. *, P < 0.05; and **, P < 0.01 calculated by ANOVA. The results represent one out of three experiments with five mice in each group. (D and G) Total and differential cell counts (D) and cytokines (G) in BAL fluid. BAL fluid was collected 24 h after challenge with intranasal OVA of the mice depicted in C. IL-25–induced pulmonary inflammation (D) and IL-13 and IL-5 production (G) were reduced in Jα18^−/− mice. The data on cytokines in G are expressed as the amounts detected in the 10-fold PBS-diluted BAL samples. Results are expressed as means ± SEM. *, P < 0.05; **, P < 0.01. The group of IL-25–treated WT mice was compared with three other groups. These results represent one out of four experiments with five mice in each group. (E and F) Histological analysis of lung tissues with hematoxylin and eosin (E) and periodic acid Schiff (F) staining. IL-25–treated WT (c) or Jα18^−/− (d) mice were compared with WT (a) or Jα18^−/− (b) mice from control (n = 4). Bars,100 μm.

Figure 4.

Requirement of IL-17RB⁺ NKT cells in the development of IL-25–induced AHR. (A) FACS profiles of NKT cells in the expression of IL-17RB. α-GalCer/CD1d dimer^hi TCRβ^hi NKT cells and α-GalCer/CD1d dimer^int TCRβ^int NKT cells in the spleen from BALB/c mice were gated, and IL-17RB expression was analyzed using F(ab′)₂ IL-17RB mAb B5F6. IL-17RB was preferentially expressed in α-GalCer/CD1d dimer^hi TCRβ^hi NKT cells (n = 3). (B) FACS profiles of NKT cells in mice treated with IL-17RB mAb. α-GalCer/CD1d dimer⁺ TCRβ⁺ NKT cells in the spleen from BALB/c mice at the indicated days after injection of 3H8 IL-17RB mAb or control rat IgG2a (1 mg/mouse) were gated and analyzed by FACS. Depletion of IL-17RB⁺ NKT cells persisted for at least 5 d after 3H8 injection because of the absence of α-GalCer/CD1d dimer^hi TCRβ^hi NKT cells in A (n = 2). Percentages are shown. (C) Development of AHR in mice treated with IL-17RB mAb. The detailed method for development of OVA/IL-25–induced AHR is described in Materials and methods. In brief, OVA/alum-sensitized mice were injected with 3H8 IL-17RB mAb or rat IgG2a (1 mg/mouse) 24 h before 2 μg IL-25 or PBS treatment and were challenged with OVA after 24 h, and R_L was determined after 48 h. The group of rat IgG2a–treated WT mice was compared with three other groups. Results are expressed as means ± SEM. The results represent one out of three experiments with five mice in each group. *, P < 0.05; and **, P < 0.01 calculated by ANOVA. (D) AHR development after cell transfer of spleen IL-17RB⁺ NKT cells into Jα18^−/− mice. The detailed method for development of OVA/IL-25–induced AHR is described in Materials and methods. Indicated cell numbers of sorted IL-17RB⁺, IL-17RB⁻ NKT, or total NKT cells from spleen or PBS control were intravenously transferred into OVA/alum-sensitized Jα18^−/− mice 24 h before treatment with 3 μg IL-25 and were challenged with OVA after 24 h, and R_L was measured after 48 h. Each group of IL-17RB⁺ NKT cell–transferred mice was compared with three other groups. *, P < 0.05; and **, P < 0.01 calculated by the Kruskal-Wallis test. The results represent one out of four experiments with five mice in each group. Results are expressed as means ± SEM.