T2 and T17 cytokines alter the cargo and function of airway epithelium-derived extracellular vesicles

Abstract

Background: Asthma is a common and heterogeneous disease that includes subgroups characterized by type 2 (T2) or type 17 (T17) immune responses for which there is a need to identify the underlying mechanisms and biomarkers in order to develop specific therapies. These subgroups can be defined by airway epithelium gene signatures and the airway epithelium has also been implicated to play a significant role in asthma pathology. Extracellular vesicles (EVs) carry functional biomolecules and participate in cell-to-cell communication in both health and disease, properties that are likely to be involved in airway diseases such as asthma. The aim of this study was to identify stimulus-specific proteins and functionality of bronchial epithelium-derived EVs following stimulation with T2 or T17 cytokines.

Methods: EVs from cytokine-stimulated (T2: IL-4 + IL-13 or T17: IL-17A + TNFα) human bronchial epithelial cells cultured at air-liquid interface (HBEC-ALI) were isolated by density cushion centrifugation and size exclusion chromatography and characterized with Western blotting and electron microscopy. Transcriptomic (cells) and proteomic (EVs) profiling was also performed.

Results: Our data shows that EVs are secreted and can be isolated from the apical side of HBEC-ALI and that cytokine stimulation increases EV release. Genes upregulated in cells stimulated with T2 or T17 cytokines were increased also on protein level in the EVs. Proteins found in T17-derived EVs were suggested to be involved in pathways related to neutrophil movement which was supported by assessing neutrophil chemotaxis ex vivo.

Conclusions: Together, the results suggest that epithelial EVs are involved in airway inflammation and that the EV proteome may be used for discovery of disease-specific mechanisms and signatures which may enable a precision medicine approach to the treatment of asthma.

Keywords: Asthma; Exosomes; Mediators of inflammation; Proteomics; Respiratory epithelium.

Fig. 1

Schematic overview of the experimental workflow. Human bronchial epithelial cells were cultured at air-liquid interface, stimulated with T2 (IL-4 and IL-13) or T17 (IL-17A + TNFα) cytokines or left untreated. Cellular gene expression was analyzed, extracellular vesicles were isolated, and the characteristics and protein cargo of the extracellular vesicles released during the different conditions were evaluated

Fig. 2

T2 and T17 cytokines induce increased release of extracellular vesicles from HBECs at air-liquid interface. Cells were stimulated with T2 (IL-4 + IL-13) or T17 (IL-17A + TNFα) cytokines or left untreated (Control) and vesicles were isolated from the apical side. a Number of particles was measured in each fraction of the size exclusion chromatography by nanoparticle tracking analysis. (n = 3) Data are presented as the mean and SEM. b Number of particles was measured in each pool, each consisting of six fractions from the size exclusion chromatography, by nanoparticle tracking analysis. (n = 3) Data are presented as the mean and SEM. c Presence of the extracellular vesicle marker flotillin-1 and the endoplasmic reticulum protein calnexin were determined by Western blot in all pools. d Size and morphology of vesicles was determined by electron microscopy. Scale bars are 200 nm in the electron micrographs. e Individual particle concentrations for the pools consisting of fractions 7–12 from b are plotted. p-values calculated using paired t-test

Fig. 3

T2 and T17 cytokines induce specific gene expression signatures in HBECs at air-liquid interface. a-b Next-generation sequencing was used to analyze RNA isolated from a single donor after 24 h stimulation. Top 20 upregulated genes after T2 (IL-4 + IL-13, a) and T17 (IL-17A + TNFα, b) stimulation. c Validation of a selection of upregulated genes by quantitative PCR (n = 5, each column represents expression in one donor). All data are expressed as log2 fold change compared to non-stimulated control

Fig. 4

GO Terms associated with the proteome of bronchial epithelium-derived extracellular vesicles. Gene Ontology Term Finder was used to determine the most enriched cellular compartments (a), biological processes (b) and KEGG pathways (c) associated with proteins detected by mass spectrometry in isolated vesicles, compared to the genome frequency. The 15 most enriched terms (based on p-value) in each category are displayed

Fig. 5

The proteome of bronchial epithelium-derived extracellular vesicles is altered upon T2 and T17 stimulation. Quantitative proteomics (tandem mass tag; TMT) was used to determine the influence of T2 and T17 cytokines on bronchial epithelium-derived extracellular vesicles. a Principle component analysis illustrating the relationship between T2-derived EVs (red), T17-derived EVs (green) and EVs isolated under non-stimulated condition (blue) (n = 3). b-c) Volcano plots of the proteome after T2 (b) and T17 (c) stimulation. Dotted lines indicate cut offs, which is 1.3 on the Y-axis (corresponding to p < 0.05) and 0.67 on the X-axis (corresponding to fold change> 1.5). d-e) Top 15 upregulated proteins in extracellular vesicles derived from bronchial epithelial cells after T2 (d) and T17 (e) stimulation based on fold change compared to non-stimulated cells f-g) Top 10 downregulated proteins in extracellular vesicles derived from bronchial epithelial cells after T2 (f) and T17 (g) stimulation based on fold change compared to non-stimulated cells

Fig. 6

The altered T17 EV proteome contains proteins with activating effects on neutrophil migration. a-b Differential levels of proteins involved in the pathways related to neutrophil migration identified in Table 2 that were predicted to be activated by T17-EV proteins (a) or inhibited by T2-EV proteins (b). c Peripheral blood neutrophils allowed to migrate towards; media only as negative control, EVs (30 μg/ml) from non-stimulated epithelial cells (blue) or cells stimulated with T2 (red) or T17 (green) cytokines, or the positive control (5% FBS). Each dot represents the median number of migrated cells after 3 h of incubation (2–4 replicate wells) for one donor, with group means ± SD indicated by lines. (n = 7–8) Group means were compared using mixed-effects analysis. Only adjusted p-values ≤0.05 after Tukey’s multiple comparisons test are shown (*: p ≤ 0.05, **: p ≤ 0.01)