Programmed cell death ligand 2 regulates TH9 differentiation and induction of chronic airway hyperreactivity

Abstract

Background: Asthma is defined as a chronic inflammatory disease of the airways; however, the underlying physiologic and immunologic processes are not fully understood.

Objective: The aim of this study was to determine whether TH9 cells develop in vivo in a model of chronic airway hyperreactivity (AHR) and what factors control this development.

Method: We have developed a novel chronic allergen exposure model using the clinically relevant antigen Aspergillus fumigatus to determine the time kinetics of TH9 development in vivo.

Results: TH9 cells were detectable in the lungs after chronic allergen exposure. The number of TH9 cells directly correlated with the severity of AHR, and anti-IL-9 treatment decreased airway inflammation. Moreover, we have identified programmed cell death ligand (PD-L) 2 as a negative regulator of TH9 cell differentiation. Lack of PD-L2 was associated with significantly increased TGF-β and IL-1α levels in the lungs, enhanced pulmonary TH9 differentiation, and higher morbidity in the sensitized mice.

Conclusion: Our findings suggest that PD-L2 plays a pivotal role in the regulation of TH9 cell development in chronic AHR, providing novel strategies for modulating adaptive immunity during chronic allergic responses.

FIG 1

Development of pulmonary T_H9 cells after chronic exposure to Aspergillus fumigatus (A.f.). A, Protocol of immunization to induce chronic AHR by A fumigatus. Briefly, a group of naive BALB/c mice were immunized intranasally (i.n.) for 46 days with A fumigatus or PBS. B, On days 25, 32, 39, and 46, the percentages of cytokines (IL-9, IL-4, IL-13, IL-10, and IL-17a) secreting Teff cells in the lungs were then assessed by means of intracellular staining gated on the CD45⁺CD3⁺CD44⁺CD4⁺ population. Data are representative of 4 independent experiments (n = 5), with P values of less than .001 for IL-9 on days 39 and 46 between PBS- and A fumigatus–treated mice.

FIG 2

PD-L2 regulates T_H9 cell development in vitro. Purified CD4⁺DO11.10⁺ T cells were put in culture (1×10⁵ cells per well) with OVA-loaded bone marrow–derived DCs (BM-DC) from WT and PD-L2^−/− mice in the presence of IL-4 (10 ng/mL), TGF-β (1 ng/mL), and anti–IFN-γ antibodies (10µg/mL) with or without anti–PD-1 blocking (α-PD1), anti–PD-L2 blocking (α-PD-L2), or isotype control antibodies (10 µg/mL). After 3 days of culture, production of IL-9, IL-1α, and IL-2 was measured by means of ELISA in the supernatant. Data are means ± SEMs of 3 experiments. *P < .05,WT isotype–treated versus WT anti–PD-L2–treated or PD-L2^−/− mice.

FIG 3

PD-L2 regulates T_H9 cell differentiation in vivo. A, Protocol of immunization to induce chronic AHR by A fumigatus (A.f.). B, Purified lung CD45⁺ cells were cultured 24 hours after the last intranasal (i.n.) immunization overnight without any stimulation and analyzed for the percentage of cytokine-secreting Teff cells by means of intracellular staining gated on the CD45⁺CD3⁺CD44⁺CD4⁺ cells. Statistical analyses were performed by using the Student t test: *P <s .05. Values are representative of 3 independent experiments (n = 5).

FIG 4

Increased airway inflammation and AHR in PD-L2^−/− mice. A, Groups of WT and PD-L2^−/− BALB/c mice were immunized intranasally with A fumigatus (A.f.) or PBS, as described Fig 1, A. The mice were then assessed for AHR 24 hours after the last intranasal immunization by measuring R_L and C_dyn. Data are means ± SEMs and representative of 3 separate experiments (n = 5). *P < .05 and **P < .03, Student t test. B, Bronchoalveolar lavage (BAL) fluid from the mice in Fig 4, A, was analyzed 24 hours after AHR measurement. Results are shown as the total number of cells in BAL fluid. *P < .05 and **P < .01, Student t test. Eos, Eosinophils; Lym, lymphocytes; Mac, monocyte/macrophage; PMN, neutrophils; Total, total cell number. C, Lung tissue from A fumigatus–sensitized PD-L2^−/− or WT mice were stained with hematoxylin and eosin (H&E; upper panel) and analyzed for cell infiltration. Lung tissue from the same mice were stained with periodic acid–Schiff (PAS; lower panel) and analyzed for the presence of mucus. Arrows indicate the production of mucus in the lumen. Original magnification ×40 (inset, magnification ×100). D, Quantifications of lung histopathology shown as thickness of airway epithelium (left panel), number of inflammatory cells per 250 µm² of lung tissue (middle panel), and number of goblet cells per 50 µm of epithelium at baseline (right panel). Data are shown as means ± SEMs (n = 5). *P < .05.

FIG 5

Variation of TGF-β production in lungs of PD-L2^−/− mice. A, Protocol for A fumigatus (A.f.) immunization and preparation of lung lysate samples from WT and PD-L2^−/− BALB/c mice. i.n., Intranasal. B, Concentrations of IL-9 and TGF-β, IL-13, IL-10, IL-4, IL-1α, and IL-17a were then assessed by means of ELISA in the whole-lung lysates, as described in the Methods section. Statistical analyses were performed with the Student t test. *P < .05 and **P < .01. Values are representative of 3 separate experiments (n = 5).

FIG 6

IL-9 regulates the severity of chronic AHR. A, Protocol of immunization of WT and PD-L2^−/− mice with A fumigatus (A.f.) and antibodies. i.n., Intranasal. B, A group of WT and PD-L2^−/− BALB/c mice were immunized intranasally according to our protocol in Fig 6, A. Twenty-four hours after the last immunization (day 46), mice were assessed for the development of AHR by measuring R_L and C_dyn. Data are means ± SEMs and representative of 3 experiments (n = 5). *P < .05 and **P < .01, Student t test. C, Lung tissue from A fumigatus (A.f.)–sensitized PD-L2^−/− or WT mice were stained with hematoxylin and eosin (H&E; upper panel) and periodic acid–Schiff (PAS; lower panel) and analyzed for cell infiltration and mucus production, respectively. Arrows indicate the production of mucus in the lumen. Original magnification ×40 (inset, original magnification ×100). D, Quantifications of lung histopathology shown as thickness of airway epithelium (left panel), number of inflammatory cells per 250 µm² of lung tissue (middle panel), and number of goblet cells per 50 µm of epithelium at baseline (right panel). Data are shown as means ± SEMs (n = 5). *P < .05. E, Survival curves of PD-L2^−/− and WT mice immunized as described in Fig 6, A. **P < .01, as determined by using a Mantel Cox test. Values are representative of 3 independent experiments starting with 10 mice per group. Ab, Antibody.