Induction of airway hyperreactivity by IL-25 is dependent on a subset of invariant NKT cells expressing IL-17RB

Abstract

IL-25 has been shown to induce Th2 responses and airway hyperreactivity (AHR) in mice, but the mechanism of action is not understood and it is unclear which cells mediate this disease. In this study we show that the receptor for IL-25, IL-17RB, is highly expressed on a subset of naive and activated CD4(+) invariant NKT (iNKT) cells, but not on activated T cells. IL-17RB(+) iNKT cells produced large amounts of Th2 cytokines that were substantially increased by IL-25 stimulation. Furthermore, IL-17RB(+) iNKT cells were capable of restoring AHR in iNKT cell-deficient mice, whereas IL-17RB(-) iNKT cells failed to reconstitute AHR and lung inflammation. Finally, IL-17RB(+) iNKT cells were detected in the lungs of wild-type mice, and induction of AHR by intranasal administration of IL-25 was significantly impaired in iNKT cell-deficient mice. Overall, our data suggest a critical role for iNKT cells in IL-25-mediated AHR. These results may lead to novel therapeutic approaches to target IL-17RB(+) iNKT cells for the treatment of allergic asthma.

FIGURE 1

A subset of CD4⁺ iNKT cell expresses IL-17RB. iNKT cells were positively selected with a CD1d tetramer loaded with α-GalCer as described in Materials and Methods. iNKT cell subsets (25 × 10³) were cultured with 1 × 10³ BMDCs as described previously (24). a, Cells were stained with IL-17RB mAb and analyzed by flow cytometry gated on enriched CD4⁺ or DN iNKT cells. IL-17RB⁺ cells were quantified and the percentages of IL-17RB⁺CD4⁺ iNKT cells are indicated in the histograms. b, CD4⁺ and DN iNKT cells were enriched from the spleens of BALB/c mice as described in Materials and Methods. CD4⁺ or DN iNKT cells (25 × 10³) were cultured with 10³ BMDCs. Cells were collected at various time points after cell culture and analyzed for the expression of IL-17RB by quantitative real-time PCR. Also, 25 × 10³ positively selected iNKT cells from the lungs of BALB/c mice with AHR were cultured with 10³ BMDCs for 48 h and analyzed for the expression of IL-17RB. As a negative control, 1 × 10⁶ BMDCs were also cultured for 48 h and analyzed for IL-17RB expression. Data are representative of three separate experiments. The IL-17RB expression levels in CD4⁺ and lung iNKT cells were compared with those in the appropriate DN iNKT cells *, p < 0.01.

FIGURE 2

IL-17RB⁺ iNKT cells preferentially produce Th2 cytokines. IL-17RB⁺ or IL-17RB⁻ iNKT cells were sorted as described in Materials and Methods. Cells were incubated with α-GalCer-loaded BMDCs in the presence or absence of IL-25 (50 ng/ml). Supernatants were collected after 48 h and cytokines were measured by ELISA. ELISA data are representative of three separate experiments and are shown as mean ± SD for triplicate samples. The cytokine levels from IL-25-treated IL-17RB⁺ cells were compared with IL-25-treated IL-17RB⁻ cells; *, p < 0.01.

FIGURE 3

IL-17RB⁺ iNKT cells restore airway hyperreactivity in Jα18^−/− mice. a, IL-17RB⁺ or IL-17RB⁻ iNKT cells were positively selected from the spleens of BALB/c mice and adoptively transferred (2 × 10⁶ cells) into OVA-sensitized Jα18^−/− mice that were then challenged with OVA (50 µg) i.n. on three consecutive days and assessed for AHR by measuring Penh (left panel), lung resistance (R_L; middle panel), and dynamic compliance (C_dyn; right panel). As controls, nonrecipient WT BALB/c or Jα18^−/− mice were sensitized (i.p.) and challenged (i.n.) with OVA. Data are the mean ± SD and are representative of three experiments (n = 5) with the following p values: *, p < 0.01; and **, p < 0.001 (recipients of IL-17RB⁺ vs recipients of IL-17RB⁻ iNKT cells). b, Lung histopathology of Jα18^−/− recipient mice from a. Lung tissue from an OVA-challenged Jα18^−/− mouse shows normal airway and surrounding parenchyma (far right panel). Lung tissue from an OVA-treated Jα18^−/− mouse that received 2 × 10⁶ wild-type iNKT cells shows inflammatory cells surrounding the airways (second panel from left). Lung parenchyma of an OVA-sensitized Jα18^−/− mouse that received 2 × 10⁶ iNKT cells from IL-17RB^−/− mice shows minimal mucus production and negligible cellular infiltration (third panel from left). However, lung tissue from OVA-treated Jα18^−/− mice that received 2×10⁶ IL-17RB⁺ iNKT cells, shows a significantly higher infiltration of inflammatory cells and mucus production (far left panel). All stainings are H&E (original magnification, ×200).

FIGURE 4

iNKT cell-deficient mice are resistant to the induction of AHR by IL-25. a, Naive BALB/c, Jα18^−/−, or CD1d^−/− mice were treated with three consecutive doses of IL-25 (0.75 µg per dose) i.n. (day s1, 2, and 3). AHR was assessed by measuring Penh (left panel), lung resistance (R_L; middle panel), and dynamic compliance (C_dyn; right panel) on day 4. As controls, mice were sensitized (i.p.) and challenged (i.n.) with OVA. Data are the mean ± SD and are representative of three experiments (n = 4) with the following p values: *, p < 0.01; and **, p < 0.001 (BALB/c plus IL-25 vs Jα18^−/− plus IL-25 or CD1d^−/− plus IL-25). b, BAL fluid from the mice in a was analyzed 24 h after AHR measurement. Results are shown as the number of cells per milliliter in BAL fluid. Eos, eosinophils; Lym, lymphocytes; Mo, monocytes/macrophages; TCC, total cell count. The total cell number and the eosinophils from the BAL of WT mice treated with OVA or IL-25 were compared with Jα18^−/− or CD1d^−/− mice treated with OVA or IL-25; *, p < 0.01; and **, p < 0.001.

FIGURE 5

Pulmonary iNKT cells express high levels of IL-17RB. Lymphoid cells purified from the lungs of BALB/c mice that had been immunized and challenged with OVA or IL-25 to induce AHR were stained with α-GalCer-loaded, PE-conjugated CD1d tetramers and anti-TCR Vβ after gating on total CD45⁺ cells. IL-17RB expression was analyzed by gating on a CD1d tetramer and TCR Vβ-positive iNKT cells. IL-17RB⁺ cells were quantified and the percentages of IL-17RB⁺ iNKT cells are indicated in the histograms. Filled histograms represent the isotype control.