Aspirin-Exacerbated Respiratory Disease Involves a Cysteinyl Leukotriene-Driven IL-33-Mediated Mast Cell Activation Pathway

Abstract

Aspirin-exacerbated respiratory disease (AERD), a severe eosinophilic inflammatory disorder of the airways, involves overproduction of cysteinyl leukotrienes (cysLTs), activation of airway mast cells (MCs), and bronchoconstriction in response to nonselective cyclooxygenase inhibitors that deplete homeostatic PGE2. The mechanistic basis for MC activation in this disorder is unknown. We now demonstrate that patients with AERD have markedly increased epithelial expression of the alarmin-like cytokine IL-33 in nasal polyps, as compared with polyps from aspirin-tolerant control subjects. The murine model of AERD, generated by dust mite priming of mice lacking microsomal PGE2 synthase (ptges(-/-) mice), shows a similar upregulation of IL-33 protein in the airway epithelium, along with marked eosinophilic bronchovascular inflammation. Deletion of leukotriene C4 synthase, the terminal enzyme needed to generate cysLTs, eliminates the increased IL-33 content of the ptges(-/-) lungs and sharply reduces pulmonary eosinophilia and basal secretion of MC products. Challenges of dust mite-primed ptges(-/-) mice with lysine aspirin induce IL-33-dependent MC activation and bronchoconstriction. Thus, IL-33 is a component of a cysLT-driven innate type 2 immune response that drives pathogenic MC activation and contributes substantially to AERD pathogenesis.

Figure 1. Expression of IL-33 protein in nasal polyps

A. Western blotting of nasal polyp proteins from 4 subjects with AERD and 4 CHES controls showing full-length (30 kDa) and processed (25, 18–21 kDa) forms of IL-33. B. Quantitative densitometry of full-length and 18–21 kDa IL-33 signals, corrected for GAPDH. The densitometry results are from 8 samples per clinical group, including the 4 from each group depicted in A. C. Tyramide-amplified anti-IL-33 immunofluorescent staining of nasal polyps from subjects with AERD and with AT chronic hyperplastic eosinophilic sinusitis. Specific immunofluorescence is shown in cells localizing to the basal layer of the epithelium (short arrows). Nonspecific staining with tyramide (larger arrows) appears in both specimens. * p<0.05, *** p<0.001.

Figure 2. Expression of IL-33 protein in the lungs of AERD-like mice

A. ELISA measurement of IL-33 in lysates of whole lung from the indicated groups. B. Western blot showing full-length and cleaved IL-33 protein in the lungs collected from WT and ptges^−/− mice 24 h after the last of 6 intranasal doses of either PBS or Df. C. Immunohistochemical staining for IL-33 protein (left) or isotype control (right) in the lungs of Df-treated ptges^−/− mice. Epithelial staining is indicated by arrows. Results in A–C are representative of at least 10 mice in two separate experiments. * p<0.05, ** p<0.01,

Figure 3. Effect of LTC₄S deletion on IL-33 expression and attendant physiologic effects

A. BAL fluid total cell counts and eosinophil counts in the lungs of ptges^−/− and ptges^−/−/ltc4s^−/− mice measured 24 h after the last of 6 doses of PBS or Df. B. ELISA of total IL-33 protein levels in lung lysates from the indicated strains. C. Western blotting of lung proteins from the indicated strains showing full-length and cleaved forms of IL-33. D. Quantitative densitometry showing effect of ltc4s deletion from ptges^−/− mice on lung IL-33 protein levels. E. BAL fluid levels of MC granule-derived mediators from the indicated groups. Results in A, B, and D and E are mean ± SEM from at least 10 mice/group in two separate experiments. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4. Role of IL-33 in Lys-ASA-induced changes in lung function and MC activation

A. Effect of blocking IL-33 or ST2 on pulmonary responses to Lys-ASA challenge. Ptges^−/− mice were challenged with Lys-ASA by inhalation 24 h after the last of 6 intranasal doses of Df. R_L was monitored continuously for 45 min and the peak change from baseline was recorded for each mouse. The indicated groups received single intraperitoneal doses of rat anti-mouse IL-33, IgG isotype control, recombinant ST2-Fc fusion protein, or Fc control. B. Levels of BAL fluid mMCP-1, histamine, and PGD₂ in BAL fluids from the same mice. Results are from 10 mice/group in two separate experiments. * p<0.05, ** p<0.01, *** p<0.001.

Figure 5. Effect of exogenous cysLTs on airway physiology and intrapulmonary MC activation

A. Peak changes in R_L in Df-treated mice from the indicated genotypes challenged by inhalation with Lys-ASA or with LTC₄, LTD₄, or LTE₄. B. Levels of mediators in BAL fluids collected 45 min after challenges with Lys-ASA or the indicated cysLT. Results in A and B are from 10 mice/group in two separate experiments. * p<0.05, ** p<0.01, *** p<0.001.

Figure 6

Effect of anti-IL-33 and ST2 blockade on pulmonary responses to direct challenges with LTE₄. The indicated groups were treated single intraperitoneal doses of rat anti-mouse IL-33, IgG isotype control, recombinant ST2-Fc fusion protein, or Fc control 24 h before the challenge with LTE₄. A. Peak percent changes in R_L from baseline are displayed. B. BAL fluid content of histamine, mMCP-1, and PGD₂. C. Effects of CysLT₁R blockade or TP receptor antagonism on LTE₄-induced changes in R_L The indicated groups received Montelukast P.O. in drinking water beginning 24 h before challenge, or received a single intraperitoneal dose of the selective TP receptor antagonist SQ29,548. * p<0.05, ** p<0.01, *** p<0.001.