Dectin-2 sensing of house dust mite is critical for the initiation of airway inflammation

Abstract

How the immune system senses aeroallergens and triggers an aberrant inflammation is poorly understood. Dectin-2 is a house dust mite (HDM)-sensing pattern recognition receptor. In a 3-week mouse model of repeated intranasal HDM challenge, anti-Dectin-2 potently attenuated the characteristic allergic inflammation and airway hyper-responsiveness. Anti-Dectin-2 also prevented neutrophil influx following a single HDM challenge. Interestingly, cysteinyl leukotrienes, but not chemokine and cytokine levels were inhibited by anti-Dectin-2 in this acute model, and in ex vivo challenge of cultured alveolar macrophages with HDM. Furthermore in the single-challenge model, zileuton, an inhibitor of leukotriene production, produced a similar effect as Dectin-2 blockade. Together these data suggest alveolar macrophage sensing of HDM by Dectin-2 elicits the production of cysteinyl leukotrienes, and this axis is key for the initiation of airway inflammation to this aeroallergen. Finally, we found Dectin-2-positive infiltrating cells present in bronchial biopsies from asthmatic subjects.

Figure 1

Neutralisation of Dectin-2 before house dust mite (HDM) allergen challenge ablates airway inflammation and airway hyper-responsiveness (AHR). Mice were treated with phosphate-buffered saline (PBS), anti-Dectin-2 or isotype control 1 day before and throughout the 3-week chronic HDM model or with prednisone for the final 2 weeks. Total airways resistance was determined in response to increasing concentrations of methacholine, as a measure of AHR (a). Cellular recruitment to the airway lumen was determined by bronchoalveolar lavage, followed by differential counting of total cells (b), eosinophils (c) and neutrophils (d). Histology was performed using hematoxylin and eosin staining on formalin-fixed lung parenchyma (f–j; bar=500 μm), and assessed by a semi-quantitative scoring system (0–5) (e). Lungs were homogenized and assayed for cytokine content by Meso-Scale Discovery or enzyme-linked immunosorbent assay. Levels were normalized to mean values from HDM/PBS-treated animals for each independent experiment (k–m). With the exception of e (median±s.e.m.; range n=6–10), data are expressed as mean±s.e.m., n=18–30 mice per group and are a composite of three independent experiments. Significance was determined by 1-way analysis of variance (ANOVA) comparing all groups, but from untreated animals to all groups not shown. For a, 2-way ANOVA was used, and significance to HDM/PBS shown. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Dectin-2 is redundant in established allergic inflammation induced by house dust mite (HDM). Mice were sensitized and challenged as per the HDM chronic model with anti-Dectin-2 or isotype control antibody dosed only during the final 2 weeks. Total airways resistance was determined in response to increasing concentrations of methacholine, as a measure of airway hyper-responsiveness (a). Cellular recruitment to the airway lumen was determined by bronchoalveolar lavage, followed by differential counting of total cells (b), eosinophils (c) and neutrophils (d). Data are expressed as mean±s.e.m., n=18–30 mice per group and are a composite of three independent experiments. Significance was determined by 1-way analysis of variance (ANOVA) comparing all groups except a, where a 2-way ANOVA was used and only significance to HDM/PBS shown. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Neutrophil recruitment, but not cytokine or chemokine production, is Dectin-2 dependent in response to a single house dust mite (HDM) challenge. Mice were treated with anti-Dectin-2 or isotype control 1 day before a single HDM challenge. Cellular recruitment to the airway lumen was determined by bronchoalveolar lavage, followed by differential counting 24 h following challenge (a). BALF was assayed for cytokine content by Meso-Scale Discovery, and levels were normalized to mean values from HDM/PBS-treated animals for each independent experiment (b–c). (d) Cysteinyl leuokitrene levels were determined by in BALF 2 h following HDM challenge. Data are expressed as mean±s.e.m., n=7–12 mice per group and are a composite of two independent experiments. Significance was determined by 1-way analysis of variance comparing all groups. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

Dectin-2 regulates house dust mite (HDM)-induced cysteinyl leukotriene but not cytokine responses in primary murine alveolar macrophages. BALF cells from unchallenged mice (>95% alveolar macrophages) were plated overnight in GM-CSF (10 ng ml⁻¹), and then pre-treated with antibodies (10 μg ml⁻¹) 1 h before HDM (10 μg ml⁻¹) stimulation. Cysteinyl leukotriene generation was assessed at 30 min (a) and cytokine induction after 24 h (b–d). Data are expressed as mean±s.e.m., n=3 performed in triplicate. Significance was determined by 1-way analysis of variance comparing all groups. *P<0.05, **P<0.01.

Figure 5

The synthesis of leukotrienes is required for the innate sensing of house dust mite (HDM). Mice were treated with the 5-lipoxygenase inhibitor zileuton (10 mg kg⁻¹; p.o.) or vehicle (0.5% methylcellulose containing 0.2% Tween 80; p.o.) 1 h before, 3 and 7 h post HDM challenge. Cellular recruitment to the airway lumen was determined by bronchoalveolar lavage, followed by differential counting (a). BALF was assayed for cytokine content by Meso-Scale Discovery (b, c). Data are expressed as mean±s.e.m., n=6–8 mice per group, and significance was determined by 1-way analysis of variance comparing all groups. **P<0.01, ***P<0.001.

Figure 6

Dectin-2 expression in bronchial biopsies from non-smoking healthy and asthmatic patients. Anti-Dectin-2 (a,b) or rabbit IgG control (c,d) immunohisotchemical staining of brochial biopsies from a non-smoking healthy (a,c) or GINA 2 asthmatic patient (b,d). Typical areas of laminar propria are shown, and bar represents 50 μm. (e) Quantification of Dectin-2-positive cells per area of lamina propria in bronchial biopsies from healthy controls (●) or asthmatic patients (X GINA1; ▪ GINA2; ▾ GINA3; ▴ GINA4). Mean±s.e.m. are shown for healthy controls and total asthmatic patients. Open symbols represent quantification with control IgG.