Nasal Epithelial Extracellular Vesicles Correlate with Type 2 Inflammation during Aspirin-induced Respiratory Reactions

Abstract

Extracellular vesicles (EVs) are membrane-bound particles secreted by cells with emerging roles in intercellular communication during tissue homeostasis and disease. Although EVs are abundant in respiratory biofluids, their cellular sources, critical cargos, and functions in the airway remain poorly understood. To determine how EV populations are changed in respiratory fluids during a chronic tissue inflammatory response, nasal EVs were assayed in 23 control participants and 22 participants with aspirin-exacerbated respiratory disease (AERD). Nasal lining fluid from participants was found to contain abundant EVs by electron microscopy and tunable resistive pulse sensing. Subset-specific EV subpopulations defined by the macrophage marker CD14 or the epithelial marker CD133/1 were increased in participants with AERD. To test how EVs change during an acute exacerbation, nasal lining fluid EVs were assessed in participants with AERD, who were repeatedly sampled during an aspirin-induced respiratory reaction. The abundance of several EV subpopulations dynamically correlated with concentrations of cysteinyl leukotrienes and tryptase in AERD nasal lining fluid. Together, these data implicate EVs in a dynamic signaling network that drives tissue inflammation during aspirin-induced type 2 immune activation in AERD.

Keywords: aspirin-exacerbated respiratory disease; chronic rhinosinusitis; extracellular vesicles; leukotrienes; nasal lining fluid.

Figure 1.

Extracellular vesicles (EVs) are present in the nasal lining fluid. (A and B) Transmission EM of SEC-purified EVs from control (A) and aspirin-exacerbated respiratory disease (AERD) (B) nasal fluid. n = 1 pooled sample of three control or AERD samples. Scale bars: 600 nm; (inset) 200 nm. (C–E) Concentration (C) and mean (D) and median (E) diameter of EVs in control and AERD nasal fluid measured using tunable resistive pulse sensing. n = 23 control samples, n = 15 AERD samples, Mann-Whitney test. (F) Schematic of bead-based EV flow cytometry assay. EVs are bound to capture beads by antibodies to surface proteins and then detected by APC-conjugated antibodies against CD9 and CD63. (G) Tetraspanin abundance on EVs from control and AERD nasal fluid, measured using bead-based EV flow cytometry. n = 23 control samples, n = 22 AERD samples. (H) Tetraspanin fluorescence intensity after degradation of EVs with the detergent Triton X-100, measured using bead-based EV flow cytometry. n = 3 AERD samples. Data are presented as mean and SEM. *P ≤ 0.05. LOD = limit of detection; MESF = molecules of equivalent soluble fluorochrome; mIgG1 = mouse IgG1 antibody isotype control; PE = phycoerythrin; REA = recombinant antibody isotype control.

Figure 2.

Macrophage–derived and epithelial cell–derived EVs are present in nasal lining fluid. Heatmap showing APC fluorescence intensity for all markers included in the bead-based EV flow cytometry assay (total of 37 surface epitopes and 2 isotype controls). n = 23 control samples, n = 22 AERD samples, no clustering.

Figure 3.

CD14⁺ and CD133/1⁺ EVs are increased in AERD nasal fluid during the aspirin reaction. (A) Aspirin challenge and sampling schematic. (B) APC fluorescence intensity for markers detected above background in control participants versus participants with AERD at baseline (t = 0) and the time of peak nasal symptoms (peak TNSS). n = 23 control samples, n = 22 AERD samples, Kruskal-Wallis with Dunn’s multiple-comparison test. Data are presented as mean and SEM. *P ≤ 0.05 and **P ≤ 0.01. TNSS = total nasal symptom score.

Figure 4.

The abundance of EV subpopulations correlates with concentrations of lipid mediators and tryptase in AERD nasal lining fluid. (A–G) Spearman rho correlation values for EV cell surface markers and biomarkers in nasal fluid with P ≤ 0.05 and rho < −0.5 or >0.5 at (A–C) baseline and (D–G) during the aspirin reaction. n = 22 AERD samples. 12,13-diHOME = 12,13-dihydroxy-9Z-octadecenoic acid; 14-HDOHE =  14-hydroxydocosa-4,7,10,12,16,19-hexaenoic acid; 15-HETE = 15-hydroxyeicosatetraenoic acid; Hxb3 = hepoxilin B3; LTB4 = leukotriene B₄; LTC4 = leukotriene C₄; LTD4 = leukotriene D₄; Tetranor-PGFM = 9α,11α-dihydroxy-15-oxo-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid.

Figure 5.

Distinct cosegregation of EV tetraspanins with cell surface markers reveals EV heterogeneity in nasal fluid. Fluorescence intensity of EV cell surface markers detected when bead-bound EVs were probed with anti-CD9 and anti-CD63 separately. Dotted line represents capture isotype control. n = 8 AERD samples, Wilcoxon matched-pairs signed rank test. Data are presented as mean and SEM. *P ≤ 0.05 and **P ≤ 0.01.