Innate lymphoid cells responding to IL-33 mediate airway hyperreactivity independently of adaptive immunity

Abstract

Background: Asthma has been considered an immunologic disease mediated by T(H)2 cells and adaptive immunity. However, clinical and experimental observations suggest that additional pathways might regulate asthma, particularly in its nonallergic forms, such as asthma associated with air pollution, stress, obesity, and infection.

Objectives: Our goal was to understand T(H)2 cell-independent conditions that might lead to airway hyperreactivity (AHR), a cardinal feature of asthma.

Methods: We examined a murine model of experimental asthma in which AHR was induced with glycolipid antigens, which activate natural killer T (NKT) cells.

Results: In this model AHR developed rapidly when mice were treated with NKT cell-activating glycolipid antigens, even in the absence of conventional CD4(+) T cells. The activated NKT cells directly induced alveolar macrophages to produce IL-33, which in turn activated NKT cells, as well as natural helper cells, a newly described non-T, non-B, innate lymphoid cell type, to increase production of IL-13. Surprisingly, this glycolipid-induced AHR pathway required not only IL-13 but also IL-33 and its receptor, ST2, because it was blocked by an anti-ST2 mAb and was greatly reduced in ST2(-/-) mice. When adoptively transferred into IL-13(-/-) mice, both wild-type natural helper cells and NKT cells were sufficient for the development of glycolipid-induced AHR.

Conclusion: Because plant pollens, house dust, and some bacteria contain glycolipids that can directly activate NKT cells, these studies suggest that AHR and asthma can fully develop or be greatly enhanced through innate immune mechanisms involving IL-33, natural helper cells, and NKT cells.

Figure 1. Blockade of IL-33 receptor, ST2, abrogates α-GalCer induced AHR

(A) Anti-mouse ST2 blocking Ab or rat IgG1_k isotype control Ab were given intravenously to the mice 24 hrs before intranasal administration of 0.5μg α-GalCer or vehicle. This data shows the mean ± SEM % of saline value and representative of three experiments. α-GalCer + anti-ST2 mAb treated group was compared with α-GalCer + isotype control mAb treated group. p<0.05 (*) and p<0.01 (**). (B) Data represent the number of cells per ml in BAL fluid. Mac, macrophage; Neu, neutrophils; Eos, eosinophils; Lymph, lymphocytes. p<0.01 (**) and p<0.001 (***). (C) 24h after intranasal α-GalCer challenge, lung tissues from each group were sectioned, and stained with hematoxylin/eosin (X40) (D) Littermate control and ST2^−/− mice were challenged with α-GalCer or vehicle, and AHR was determined by invasive measurement of airway resistance (R_L) as in (A). p<0.05 (*) (E) Number of cells in BAL fluid was counted as in (B). Mac, macrophage; Neu, neutrophils; Eos, eosinophils; Lymph, lymphocytes. p<0.05 (*) and p<0.01 (**). (F) Lung tissues from each group were stained with hematoxylin/eosin as Fig. 1C. Data are representative of at least three experiments.

Figure 2. αGalCer induces AHR by enhancing IL-33 production in the lung

(A) IL-33 mRNA was normalized to GAPDH and relative mRNA levels calculated as fold increase over vehicle control. p<0.05 (*) and p<0.001 (***). (B) Total lung homogenates were taken from vehicle or α-GalCer treated mice and IL-33 protein levels were tested by ELISA. p<0.01 (**). (C) Lung tissues from vehicle treated mice (left) or α-GalCer treated mice (right) were evaluated by IHC for IL-33 expression and immunolocalization. The higher power images on the right of each panel show type II pneumocytes, alveolar macrophages (AM), and airway epithelial cell (AEC). Original magnification: X200. (D) IL-33 expression by Type I pnemocytes (left: Aquaporin 5 positive) or Type II pnemocytes (right: surfactant protein C positive) were identified using lung tissues. The arrows indicate IL-33⁺ cells, which are surfactant protein C positive (type II pneumocytes). (E) IL-33 expression was examined by intracellular cytokine staining. Shaded: isotype control, black line: vehicle, and red line; αGalCer treatment. (F) The graph represents the total numbers of IL-33 producing cells in the lung. p<0.05 (*) and p<0.01 (**). (n≥9)

Figure 3. NKT cells induce IL-33 production from alveolar macrophages, DCs and Type II penumocytes

(A-C) Mouse NKT cells line were co-cultured with alveolar macrophages (A), DCs (B), or mouse airway epithelial cell lines (MLE12) (C). Relative mRNA levels calculated as fold increase over alveolar macrophages (A), or DCs (B). For CD1d blocking, alveolar macrophages or DCs treated with 10μg/ml of anti-CD1d mAb (HB323) 1hr before co-culture. Data present the mean ± SEM, representative of three experiments. p<0.001 (***) (D) Human NKT cells (10⁵/96 well) were co-cultured with human type II pneumocytes epithelial cell lines (A549) (5×10⁴/96 well). 10μg/ml of anti-CD1d mAb (42.1) was used for CD1d blocking experiments. Relative mRNA levels calculated as fold increase over Type II pnemocytes. p<0.05 (*) and p<0.001 (***). (E, F) The interaction between NKT cells and alveolar macrophages was blocked by anti-CD1d mAb, anti-CD40L mAb, and anti-ST2 mAb (10μg/ml). IL-33 expression was measured by mRNA expression (E) or ELISA (F). p<0.05 (*). (G, H) Alveolar macrophages (G) or DCs (H) from WT or CD1d^−/− mice co-cultured with NKT cells for 72 hrs. p<0.05 (*), p<0.01 (**) and p<0.001 (***). (I) NKT cells were placed in upper chamber and alveolar macrophages were placed in lower chamber of transwell chambers to block the NKT cells-Macrophage cell-cell contact. p<0.05 (*). (J) To inhibit the apoptosis of macrophage, 10 μg/ml of apoptosis inhibitor (Q-VD-OPH) was treated to macrophages. p<0.05 (*). Data are representative of at least three experiments.

Figure 4. Glycolipid from Sphingomonas induces AHR by producing IL-33

(A) 10μg of Sphingomonas glycolipid (PS-30) was administrated to littermate control or ST2^−/− mice. 24hr after glycolipid challenge, AHR was measured as in Fig. 1A. (n≥6) p<0.05 (*). (B) Data represent the number of cells per ml in BAL fluid and are the mean ± SEM. p<0.05 (*), p<0.01 (**) and p<0.001 (***). Mac, macrophage; Neu, neutrophils; Eos, eosinophils; Lymph, lymphocytes. (C) Lung cells were stained as in Fig 2 (D) 24hr after Sphingomonas glycolipid(PS-30) challenge. Shaded histogram, isotype control; black line, vehicle; blue line, PS-30 treated group. (D) IL-33 protein levels from lungs of mice at 24 hrs after treatment with vehicle or PS-30, measured by ELISA. p<0.05 (*). (E) BAL fluid was collected 24hr after PS-30 treatment, and NKT cells were analyzed Shaded histogram, vehicle control; black line, PS-30 exposed NKT cells. The MFI for CD25 was 147 (control, 94.7), and for CD69:1236 (control,885) Data are representative of at least three experiments (n≥6).

Figure 5. α-GalCer induces IL-13 producing natural helper cells in the lung

(A) Total lung cells were obtained from vehicle or α-GalCer treated mice, and level of IL-13 on CD45 positive cells detected by intracellular staining (upper panel). Total IL-13 positive cells were further analyzed using Abs against Lin and ST2 (lower panel). (B) Natural helper cells (lin⁻ST2⁺) subsets were gated from CD45 positive cells, and then assessed by the expression of c-Kit and Sca-1. (C) Natural helper cells (CD45⁺lin⁻ST2⁺) were gated as shown in (B), and the expression of Sca-1 and IL-13 were further analyzed. The graph represents the number of Lin⁻ ST2⁺ c-Kit⁺ Sca-1⁺IL-13⁺ natural helper cells in the lung (MFI for vehicle: 78.6±19.1, MFI for α-GalCer: 641±128.5). p<0.001(***). Data are representative of at least three experiments. (D) IL-13 producing natural helper cells from WT or ST2^−/− mice were compared as in Fig 5C. n≥4 mice in each group in this experiment.

Figure 6. IL-33 induces AHR by increasing IL-13 producing natural helper cells

(A) Recombinant IL-33 (0.1μg) administrated into WT or RAG^−/− mice for 3 consecutive days. The number of natural helper cells (CD45⁺lin⁻ST2⁺) was assessed by flow cytometry. (B) The number of IL-13 producing natural helper cells (Lin⁻ ST2⁺ c-Kit⁺ Sca-1⁺IL-13⁺) was calculated after saline or IL-33 challenge. (WT saline: 69.1±11.5×10³, WT IL-33: 1265±248.7 X10³, RAG^−/− saline:138.6±25.4×10³ and RAG^−/− IL-33:927.2±196.8×10³). p<0.05 (*). (C) WT BALB/c mice or IL-13^−/− mice were treated with 0.1μg of IL-33 for 3 consecutive days, and AHR was measured. Data represent the mean ± SEM % of saline value (n≥4). p<0.05 (*). (D) BAL fluid from mice in (C) was analyzed for airway inflammatory cells as shown in (1B). Data represent the number of cells (mean ± SEM), representative of three experiments. p<0.05 (*). Mac, macrophage; Neu, neutrophils; Eos, eosinophils; Lymph, lymphocytes.

Figure 7. Natural helper cells and NKT cells are required for α-GalCer induced AHR

(A) Schematic showing the protocol for adoptive transfer of natural helper cells or NKT cells. Change in lung resistance (B) and inflammatory cells in BAL fluid (C) in IL-13^−/− recipients (n = 4 per group) given purified natural helper cells (Lin⁻ST2⁺ subsets) from WT donors followed by vehicle or α-GalCer challenge for 24 hr. Mac, macrophage; Neu, neutrophils; Eos, eosinophils; Lymph, lymphocytes. Change in lung resistance (D) and inflammatory cells in BAL fluid (E) in IL-13^−/− recipients (n = 4 per group) given purified NKT cells from WT donors followed by vehicle or α-GalCer challenge for 24 hr. Mac, macrophage; Neu, neutrophils; Eos, eosinophils; Lymph, lymphocytes.

Figure 8. Schematic of the IL-33 – ST2 axis in the development of AHR

Upon activation by glycolipid antigens, NKT cells induce macrophages, DCs and Type II pneumocytes to produce IL-33, which in turn activates natural helper cells and NKT cells to produce IL-13, resulting in the development of AHR. IL-33 can also activate mast cells, eosinophils and basophils.