Unique Lipid Signatures of Extracellular Vesicles from the Airways of Asthmatics

Abstract

Asthma is a chronic inflammatory disease process involving the conductive airways of the human lung. The dysregulated inflammatory response in this disease process may involve multiple cell-cell interactions mediated by signaling molecules, including lipid mediators. Extracellular vesicles (EVs) are lipid membrane particles that are now recognized as critical mediators of cell-cell communication. Here, we compared the lipid composition and presence of specific lipid mediators in airway EVs purified from the bronchoalveolar lavage (BAL) fluid of healthy controls and asthmatic subjects with and without second-hand smoke (SHS) exposure. Airway exosome concentrations were increased in asthmatics, and correlated with blood eosinophilia and serum IgE levels. Frequencies of HLA-DR⁺ and CD54⁺ exosomes were also significantly higher in asthmatics. Lipidomics analysis revealed that phosphatidylglycerol, ceramide-phosphates, and ceramides were significantly reduced in exosomes from asthmatics compared to the non-exposed control groups. Sphingomyelin 34:1 was more abundant in exosomes of SHS-exposed asthmatics compared to healthy controls. Our results suggest that chronic airway inflammation may be driven by alterations in the composition of lipid mediators within airway EVs of human subjects with asthma.

Figure 1

General characteristics of BAL extracellular vesicles (EVs). (a) Electron microscopy of negative-stained BAL EVs. (b) NanoSight quantitation of a representative BAL EV isolation showing concentration on the y-axis, and size distribution on the x-axis. (c) Comparison of size distribution of EVs between each study group. (d) Comparison of EV particle concentration between healthy subjects, asthmatics, and SHS-exposed groups. Mann Whitney T test, * < 0.05. (e) Correlation of eosinophil frequency and particle concentration. Spearman’s rank correlation, * < 0.05. (f) Correlation of IgE titer and particle concentration. Spearman’s rank correlation, ** < 0.01; (Healthy Subjects, n = 9; Asthmatic Subjects, n = 11).

Figure 2

Characterization of EVs by flow cytometry. (a) ApogeeMix beads used to calibrate a BD LSRII to identify location of exosomes based on size. (b) A representative forward and side scatter of BAL EVs. (c) Frequency of CD63⁺ EVs. Mann Whitney T test, * < 0.05. (d–f) Frequency of EVs positive for HLA-DR, CD54, or CD36, after gating on CD63⁺ EVs. Mann Whitney T test, * < 0.05. (g,h) Mean fluorescence intensities of each marker analyzed on BAL EVs. Black bars represent healthy subjects (including SHS-exposed), and red bars represent asthmatics (including SHS-exposed). Mann Whitney T test, * < 0.05. (k–n) Mean fluorescence intensities of each marker analyzed on BAL EVs for each study group. Solid black bars represent healthy subjects, open black bars represent healthy subjects exposed to SHS, solid red bars represent asthmatics, and open red bars represent asthmatics exposed to SHS. Mann Whitney T test, * < 0.05; (Healthy Subjects, n = 9; Asthmatic Subjects, n = 9).

Figure 3

Heterogeneity of isolated BAL EVs. (a) The proportions of EVs which are CD9⁺CD63⁺CD81⁺TSG101⁺. Mann Whitney T test, ** < 0.01. (b) The proportions of ARF6⁻, GRP94⁻, and ARF6⁻GRP94⁻ EVs. (c) The proportions of ARF6⁻, GRP94⁻, and ARF6⁻GRP94⁻ EVs that are CD9⁺CD63⁺CD81⁻TSG101⁻. (d) The proportions of ARF6⁻, GRP94⁻, and ARF6⁻GRP94⁻ EVs that are CD9⁻CD63⁻CD81⁺TSG101⁺. Mann Whitney T test, ** < 0.01. (Healthy Subjects, n = 8; Asthmatic Subjects, n = 7).

Figure 4

Significant differences in phosphoglycerolipids and sphingolipids in BAL EVs comparing study groups. (a) The intensity of abundance of sphingomyelin 34:1 in healthy SHS-exposed subjects compared to asthmatics exposed to SHS. Mann Whitney T test, * < 0.05. Corresponding mass spectrum and proposed structure shown to the right. (b) The intensity of abundance of phosphatidylglycerol 34:2 in SHS-exposed healthy subjects and non-SHS-exposed asthmatics as compared to non-SHS-exposed healthy subjects. Mann Whitney T test, * < 0.05. Corresponding mass spectrum and proposed structure shown to the right. (c) The intensity of abundance of ceramide 34:2 in SHS-exposed and non-exposed study groups. Mann Whitney T test, * < 0.05. (d) The intensity of abundance of ceramide-phosphate 28:0 in SHS-exposed and non-exposed study groups. Mann Whitney T test, * < 0.05; (Healthy Subjects, n = 9 (n = 4, no SHS; n = 5, + SHS); Asthmatic Subjects, n = 11 (n = 6, no SHS; n = 5, + SHS)).

Figure 5

Ceramides were significantly different between healthy subjects and SHS-exposed healthy subjects. (a) Intensity of abundance of monosialoganglioside 28:3. Mann Whitney T test, * < 0.05. (b) Intensity of abundance of ceramide-phosphate 28:1. Mann Whitney T test, * < 0.05. (c) Intensity of abundance of mannosyl-diinositol phosphoryl-ceramide 26:2. Mann Whitney T test, * < 0.05; (Healthy Subjects, n = 9 (n = 4, no SHS; n = 5, + SHS); Asthmatic Subjects, n = 11 (n = 6, no SHS; n = 5, + SHS)).

Figure 6

Partial least squares (PLS) discriminant analysis of lipids extracted from EVs. (a) Comparison of lipids identified in asthmatics and healthy subjects. (b) Comparison of lipids identified in healthy subjects and healthy SHS-exposed subjects. (c) Comparison of lipids identified in asthmatics and asthmatics exposed to SHS. (d) Comparison of lipids identified in asthmatics exposed to SHS and healthy subjects exposed to SHS, (Healthy Subjects, n = 9 (n = 4, no SHS; n = 5, + SHS); Asthmatic Subjects, n = 11 (n = 6, no SHS; n = 5, + SHS)).