Dectin-2 recognition of house dust mite triggers cysteinyl leukotriene generation by dendritic cells

Abstract

House dust mites are a significant source of airborne allergen worldwide, but there is little understanding of how they so potently generate allergic inflammation. We found that extracts from the house dust mites Dermatophagoides farinae (Df) and Dermatophagoides pteronyssinus and from the mold Aspergillus fumigatus stimulated a rapid and robust production of cysteinyl leukotrienes (cys-LTs), proinflammatory lipid mediators, from mouse bone marrow-derived dendritic cells (BMDCs). Con A affinity chromatography of the Df extract revealed that the relevant ligand is a glycan(s), suggesting stimulation via a dendritic cell (DC) lectin receptor. Cys-LT production in BMDCs from wild-type mice was inhibited by spleen tyrosine kinase (Syk) inhibitors and was abolished in BMDCs from FcRgamma-/- mice, implicating either Dectin-2 or DC immunoactivating receptor. Transfection of each receptor in bone marrow-derived mast cells revealed that only Dectin-2 mediates cys-LT production by Df, Dermatophagoides pteronyssinus, and Aspergillus fumigatus. Lentiviral knockdown of Dectin-2 in BMDCs attenuated Df extract-elicited cys-LT generation, thereby identifying Dectin-2 as the receptor. Lung CD11c+ cells, but not peritoneal or alveolar macrophages, also generated cys-LTs in response to Df. These findings place Dectin-2 among the C-type lectin receptors that activate arachidonic acid metabolism and identify the Dectin-2/FcRgamma/Syk/cys-LT axis as a novel mechanism by which three potent indoor allergens may activate innate immune cells to promote allergic inflammation.

FIGURE 1

Generation of cys-LTs by BMDCs in response to Df extract. A, BMDCs from BALB/c WT or LTC₄S^−/− mice were harvested after 9 days of culture and stimulated with 100 µg/ml Df extract for the indicated times. The concentrations of cys-LTs in the supernatants were measured by enzyme immunoassay. Results are means ± SEM from four experiments. p < 0.01 compared with LTC₄S^−/−. Significance was determined using a paired Student’s t test. B, BMDCs from WT or MyD88^−/− mice were stimulated with 100 µg/ml Df extract for 60 min. Results are means ± SEM from four experiments. C and D, BMDCs from WT mice were stimulated with LPS, LPS plus OVA, or 100 µg/ml Df for 60 min (C) or with 10 µg/ml MDP, 10 µg/ml murabutide (MURA), or the calcium ionophore A23187 at 5 µM for 60 min (D). Results are means ± SEM from three experiments.

FIGURE 2

Generation of cys-LTs by pulmonary CD11c⁺ cells, alveolar macrophages, and peritoneal macrophages. Pulmonary CD11c⁺ cells (A), alveolar macrophages (B), and peritoneal macrophages (C) were harvested and stimulated by medium (−) or 100 µg/ml Df extract for 60 min. Calcium ionophore A23187 at 5 µM was used as a positive control. The concentrations of cys-LTs in the culture supernatants were measured by enzyme immunoassay. Results are means ± SEM from three experiments. ^‡, p = 0.005 compared with medium control.

FIGURE 3

Dose- and time-dependent generation of cys-LTs by BMDCs in response to extracts of Dp and Af. Day 8 BMDCs from BALB/c WT mice were stimulated with Dp (A) or Af (B) at 1, 10, and 100 µg/ml for the indicated times. The concentrations of cys-LTs in the supernatants were measured by enzyme immunoassay. Results are means ± SEM from four experiments. p = 0.001 for Dp concentration and Af concentration, p = 0.0001 for Dp and Af time-dependence. Significance was determined using two-way ANOVA.

FIGURE 4

Cys-LT generation in response to purified Df extract and inhibition by mannose. A, Df extract was separated by chloroform/methanol extraction, evaporated, and reconstituted with RPMI 1640 medium. BMDCs were stimulated with the aqueous phase (AQ) and with unfractionated Df extract for 30 min. Results are means ± SEM from three experiments. B, The aqueous phase was then subjected to Con A affinity chromatography. The bound materials were eluted sequentially with elution buffer containing 0.1, 0.2, and 0.4 M of α-methyl mannoside/α-methyl glucoside and subsequently with 10% ethanol. Fractions were evaporated, reconstituted with RPMI 1640 medium, and added to day 8 BMDCs for a 30-min stimulation. Cys-LTs in the supernatant were measured by enzyme immunoassay. Values are means ± half-range (n = 2). Results are representative of three independent experiments. C and D, BMDCs were incubated with mannose for 30 min before stimulation with Df (C) or with the 0.2 M α-methyl mannoside/α-methyl glucoside elution fraction from Con A-purified Df (D). Values are means ± SEM from three experiments.

FIGURE 5

Effect of Syk inhibition on Df, Dp, and Af extract-elicited cys-LT generation by BMDCs. Day 8 BMDCs from BALB/c WT mice were incubated with varied concentrations of a Syk inhibitor, 3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide, for 30 min at 37°C before stimulation with 100 µg/ml of either Df, Dp, or Af extract. 5 µM A23187 was used as a positive control to show the integrity of the cys-LT biosynthetic pathway. Supernatants were harvested 30 min after stimulation, and cys-LTs were measured by enzyme immunoassay. Results are representative of three independent experiments.

FIGURE 6

Effect of FcRγ deficiency on Df, Dp, and Af extract-elicited cys-LT generation by BMDCs. Day 8 BMDCs from BALB/c WT or FcRγ^−/− mice were stimulated with Df, Dp, or Af extract at 100 µg/ml. 5 µM A23187 was used as a positive control. Supernatants were harvested 60 min after stimulation, and cys-LTs were measured by enzyme immunoassay. Results are means ± SEM from three experiments. *, p = 0.0001; ^‡, p = 0.001 as compared with WT BMDCs.

FIGURE 7

Df, Dp, and Af extract-elicited cys-LT generation by BMMCs transfected with Dectin-2. BMMCs were transfected with FcRγ alone (A), DCAR and FcRγ (B), Dectin-2 and FcRγ (C), or Dectin-2 alone (D). Eighteen hours later, each transfectant was stained with the Dectin-2 Ab (blue), DCAR Ab (green), or isotype control Ab (black) and analyzed by flow cytometry. E, Each transfectant was stimulated with either medium or with Df, Dp, or Af extract at 100 µg/ml for 45 min, and cys-LTs in the supernatant were measured by enzyme immunoassay. Results are means ± SEM from three experiments. *, p < 0.005; ^‡, p < 0.001 as compared with BMMCs transfected with FcRγ alone.

FIGURE 8

Mannose inhibition of cys-LT generation from Dectin-2 transfectants stimulated with unfractionated Df and Con A-purified Df. A, BMMCs transfected with Dectin-2 and the FcRγ-chain were stimulated with unfractionated Df extract or the 0.2 M α-methyl mannoside/α-methyl glucoside elution fraction from Con A-purified Df (Con A-Df) for 30 min. Cys-LTs were measured in the supernatant by enzyme immunoassay. Results are means ± SEM from three experiments. **, p < 0.01 compared with medium alone. B and C, BMMCs transfected with Dectin-2 and the FcRγ-chain were incubated with mannose for 30 min before stimulation with Df (B) or Con A-Df (C). Values are means ± SEM from three experiments.

FIGURE 9

Df extract-elicited cys-LT generation in BMDCs with knockdown of Dectin-2. Day 6 BMDCs from BALB/c WT mice were harvested and infected with viral particles containing Dectin-2 shRNA or vector alone to achieve a multiplicity of infection of 5. After 72 h, Dectin-2 expression was assessed by flow cytometry. A representative histogram (A) and quantitation (B) of Dectin-2 expression on BMDCs infected with vector alone (n = 7) and BMDCs infected with viral particles containing Dectin-2 shRNA (n = 3) is shown. ^‡, p = 0.01 as compared with BMDCs infected with control virus. C, Control (n = 6) and Dectin-2 knockdown (KD, n = 3) BMDCs at 72 h were also stimulated with Df at 100 µg/ml for 60 min to assess cys-LT production. *, p = 0.004 as compared with BMDCs infected with control virus. Values in B and C are means ± SEM from three experiments.