IL-9 governs allergen-induced mast cell numbers in the lung and chronic remodeling of the airways

Abstract

Rationale: IL-9 is a pleiotropic cytokine that has multiple effects on structural as well as numerous hematopoietic cells, which are central to the pathogenesis of asthma.

Objectives: The contribution of IL-9 to asthma pathogenesis has thus far been unclear, due to conflicting reports in the literature. These earlier studies focused on the role of IL-9 in acute inflammatory models; here we have investigated the effects of IL-9 blockade during chronic allergic inflammation.

Methods: Mice were exposed to either prolonged ovalbumin or house dust mite allergen challenge to induce chronic inflammation and airway remodeling.

Measurements and main results: We found that IL-9 governs allergen-induced mast cell (MC) numbers in the lung and has pronounced effects on chronic allergic inflammation. Anti-IL-9 antibody-treated mice were protected from airway remodeling with a concomitant reduction in mature MC numbers and activation, in addition to decreased expression of the profibrotic mediators transforming growth factor-β1, vascular endothelial growth factor, and fibroblast growth factor-2 in the lung. Airway remodeling was associated with impaired lung function in the peripheral airways and this was reversed by IL-9 neutralization. In human asthmatic lung tissue, we identified MCs as the main IL-9 receptor expressing population and found them to be sources of vascular endothelial growth factor and fibroblast growth factor-2.

Conclusions: Our data suggest an important role for an IL-9-MC axis in the pathology associated with chronic asthma and demonstrate that an impact on this axis could lead to a reduction in chronic inflammation and improved lung function in patients with asthma.

Figure 1

Prolonged IL-9 neutralization throughout chronic allergen challenge is required to reduce resident mast cell (MC) numbers in the lung. Mice were sensitized and challenged according to the acute and chronic challenge protocol (Figures E1A and E1B). (A, C) MC activation was determined by levels of mMCP-1 in serum measured by ELISA. (B, D) Tracheal sections were stained with toluidine blue, and MC numbers were determined and normalized per unit area (μm²) of tissue counted. Data are expressed as mean ± SEM, n = 8–27 mice/group from one to three independent experiments, *P<0.05 or ***P<0.001, comparing groups as indicated by Mann-Whitney U test. OVA = ovalbumin.

Figure 2

IL-9 blockade significantly attenuates airway remodeling after chronic ovalbumin (OVA) challenge. Mice were sensitized and chronically challenged with allergen (Figure E1B). (A, B) Mucus production was determined from periodic acid-Schiff–stained lung sections according to criteria in Methods. (C) Total lung collagen was assessed biochemically in lung tissue homogenates, and (D) peribronchial collagen deposition was quantified by image analysis of Sirius red-stained lung sections for which representative sections of each group are shown in (E). (F) Numbers of airway smooth muscle (ASM) cells beneath the basement membrane were determined morphologically, and (G) proliferating ASM cell numbers were quantified from proliferating cell nuclear antigen-stained lung sections. Data are expressed as mean ± SEM, n = 10–27 mice/group from two to three independent experiments, *P<0.05, ***P<0.0001 comparing groups as indicated by Mann-Whitney U test.

Figure 3

IL-9 blockade is effective at preventing airway remodeling due to house dust mite (HDM) exposure. Mice were chronically challenged with intranasal HDM (Figure E1C). (A, B) Mucus production was quantified from periodic acid-Schiff–stained lung sections, according to criteria in Methods. Peribronchial collagen deposition was examined from Sirius red–stained lung sections for which representative sections of each group are shown in (C) and quantified by image analysis (D). (E, F) Tracheal sections were stained with toluidine blue and mast cells counted and normalized according to area. Data shown in A, C, and D are expressed as mean ± SEM, n = 4–6 mice/group. *P<0.05, **P<0.01 comparing groups as indicated by Mann-Whitney U test. PBS = phosphate-buffered saline.

Figure 4

Anti–IL-9 treatment reduced expression of profibrotic growth factors. (A) Transforming growth factor (TGF)β activity was measured in bronchoalveolar lavage supernatants using the secreted alkaline phosphatase bioassay as described in Methods. Levels of (B) vascular endothelial growth factor (VEGF) and (C) fibroblast growth factor (FGF)-2 were quantified in lung tissue homogenate by ELISA. Data are expressed as mean ± SEM, n = 6–18 mice/group from one to two independent experiments, *P<0.05, ***P<0.001 comparing groups as indicated by Mann-Whitney U test. OVA = ovalbumin.

Figure 5

Chronic allergen challenge is associated with impaired lung function, which is reversed by anti–IL-9 blockade. AHR was induced by chronic allergen challenge (Figure E1B) and assessed using the flexiVent system in response to increasing concentrations of methacholine. The snapshot parameters (A) resistance and (B) elastance, and the prime wave parameters (C) G (tissue resistance), and (D) H (tissue elastance) were measured. Data are expressed as mean ± SEM, n = 10–27 mice/group from three independent experiments, **P<0.01, ***P<0.001, comparing ovalbumin (OVA)-sensitized IgG-treated mice with sham-sensitized IgG-treated mice and ^#P<0.05, ^##P<0.01, ^###P<0.001 comparing OVA-sensitized anti–IL-9 treated mice with sham-sensitized anti–IL-9 treated mice by two-way analysis of variance.

Figure 6

Airway hyperresponsiveness (AHR) persists after cessation of allergen exposure. Mice were sensitized and subjected to chronic allergen challenge (Figure E1B). One month after the cessation of allergen challenge (Day 80), cellular recruitment to the airway lumen was determined by bronchoalveolar lavage, followed by (A) differential counting. AHR was measured using the flexiVent system in response to increasing concentrations of methacholine. (B) Total airways resistance (R) and (C) small airways resistance (G) were measured. Data are expressed as mean ± SEM, n = 6–8 mice/group, ***P<0.001 comparing OVA-sensitized mice with sham-sensitized mice by (A) Mann-Whitney U test or (B, C) two-way analysis of variance.

Figure 7

Human lung mast cells (MCs) express IL-9 receptor (IL-9R), and are a source of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2. (A) Tryptase⁺ MC numbers were calculated in bronchial biopsies from healthy control subjects and subjects with mild asthma. Double immunofluorescence staining of MCs and IL-9R or IL-9 was performed on paraffin sections using direct-labeled Alexa-488 anti-MC tryptase (green) and Alexa-555 detection system (red) to detect MCs and IL-9 or IL-9R, respectively. (B) Numbers of IL-9⁺ and (C) IL-9R⁺ MCs were determined in bronchial biopsies from subjects with asthma and healthy control subjects. (D) IL-9R⁺ subepithelial cells in the human airway mucosa of subjects with mild asthma, as visualized by AP/new Fuchsin detection (red) and HTX counter stain. Examples of tryptase and IL-9R colocalization within same sections are shown by (E) separate filter settings and (F) as fused images. (G–I) Examples of colocalization of MC tryptase (red) and (G) IL-9, (H) VEGF, and (I) FGF-2. Scale bars: D, E, G, I = 65 μm; F, H = 40 μm.