Thymic stromal lymphopoietin controls prostaglandin D2 generation in patients with aspirin-exacerbated respiratory disease

Abstract

Background: Prostaglandin (PG) D2 is the dominant COX product of mast cells and is an effector of aspirin-induced respiratory reactions in patients with aspirin-exacerbated respiratory disease (AERD).

Objective: We evaluated the role of the innate cytokine thymic stromal lymphopoietin (TSLP) acting on mast cells to generate PGD2 and facilitate tissue eosinophilia and nasal polyposis in patients with AERD.

Methods: Urinary eicosanoid levels were measured in aspirin-tolerant control subjects and patients with AERD. Nasal polyp specimens from patients with AERD and chronic rhinosinusitis were analyzed by using quantitative PCR, Western blotting, and immunohistochemistry. Human cord blood-and peripheral blood-derived mast cells were stimulated with TSLP in vitro to assess PGD2 generation.

Results: Urinary levels of a stable PGD2 metabolite (uPGD-M) were 2-fold higher in patients with AERD relative to those in control subjects and increased further during aspirin-induced reactions. Peak uPGD-M levels during aspirin reactions correlated with reductions in blood eosinophil counts and lung function and increases in nasal congestion. Mast cells sorted from nasal polyps expressed PGD2 synthase (hematopoietic PGD2 synthase) mRNA at higher levels than did eosinophils from the same tissue. Whole nasal polyp TSLP mRNA expression correlated strongly with mRNA encoding hematopoietic PGD2 synthase (r = .75), the mast cell-specific marker carboxypeptidase A3 (r = .74), and uPGD-M (r = 0.74). Levels of the cleaved active form of TSLP were increased in nasal polyps from patients with AERD relative to those in aspirin-tolerant control subjects. Recombinant TSLP induced PGD2 generation by cultured human mast cells.

Conclusions: Our study demonstrates that mast cell-derived PGD2 is a major effector of type 2 immune responses driven by TSLP and suggests that dysregulation of this innate system contributes significantly to the pathophysiology of AERD.

Keywords: Aspirin-exacerbated respiratory disease; Samter triad; cysteinyl leukotrienes; eosinophils; innate immunity; mast cells; nasal polyps; prostaglandin D(2); thymic stromal lymphopoietin.

FIG 1

PGD₂ production mediates effector responses in AERD. A, Baseline uPGD-M levels from healthy controls (n=9), ATA controls (n=7), and subjects with AERD (n=29). B, Surface CRTH2 expression by blood eosinophils from ATA controls and subjects with AERD. C, Correlation of surface CRTH2 expression by blood eosinophils with urinary PGD-M. D, Time-dependent changes in uPGD-M levels during reactions to oral aspirin challenges. E,F,G, Correlations of peak uPGD-M levels with changes in peripheral blood eosinophils (E), FEV₁ (F) and TNSS (G). A,D, Data are shown as means +SEM. B, Data are shown as individual points with group mean. C,E,F,G Data are shown as linear dependence measured with Pearson's correlation coefficient.

FIG 2

Expression of PGD₂ synthetic enzymes by nasal polyp effector cells. A, B, Cytofluorographic detections of mast cells (A) and eosinophils (B) in enzymatically dispersed nasal polyps. C, Toluidine blue staining of sorted nasal polyp mast cells. D, Staining of sorted nasal polyp eosinophils. E, hPGDS mRNA expression from sorted nasal polyp eosinophils and mast cells. E, Data are shown as individual points with group median as measured by Mann-Whitney test.

FIG 3

Comparison of nasal tissue mast cell counts in AERD and aspirin-tolerant CRS controls. A, Number of mast cells per mm² by immunohistochemistry in CRS and AERD. B, Percentage of CD45⁺ cells that are CD117^hi/ FcεR1⁺ in patients with CRS and AERD. A,B, Data are shown as individual points with group mean.

FIG 4

Expression of TSLP in AERD and CRS nasal polyps. A, Whole nasal polyp TSLP mRNA in CRS (n = 7) and AERD (n = 18). B, Densitometric analysis of the cleaved, active form of TSLP from patients with CRS and AERD (n = 11, both groups). C, Total protein levels of TSLP in AERD and CRS measured by Western blotting of three representative samples per group. A,B, Data are shown as mean +SEM.

FIG 5

Relationships between nasal polyp TSLP mRNA expression, mast cell markers, and systemic PGD₂ production in AERD. A, Correlation between TSLP and CPA3 mRNA expression. B, Correlation between TSLP and hPGDS mRNA expression. C, Correlation between TSLP expression and baseline uPGD-M. D, Correlation between hPGDS mRNA expression and baseline uPGD-M. A – D, Data are shown as linear dependence measured with Pearson's correlation coefficient.

FIG 6

In vitro stimulation of mast cells with TSLP. A, TSLP (10 ng/mL) increases human cord blood-derived mast cell generation of PGD₂ in vitro at 6 hours. B, TSLP (10 ng/mL) with IL-33 (10 ng/mL) synergistically increases human peripheral blood-derived mast cell generation of PGD₂ in vitro at 6 hours compared to IL-33 alone. A, B, Data are displayed for individual experiments with cells from three different donors; differences are not statistically significant.