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

Primary human bronchial epithelial cells (HBECs; Lonza, Basel, Switzerland) were expanded to passage 2, then seeded onto 0.4 μm Corning® HTS Transwell®-24 well permeable supports (Sigma-Aldrich, St Louis, MO) and differentiated at Air-Liquid Interface (ALI) using the PneumaCult™ media system (STEMCELL Technologies, Vancouver, British Columbia, Canada) according to manufacturer’s protocol.

Once fully differentiated, cells were stimulated for 24-48 h by basolateral addition of human recombinant proteins IL-4 and IL-13 or IL-17A and TNFα (all 30 ng/ml each; R&D Systems, Minneapolis, MN) diluted in PneumaCult™-ALI medium.

Cells stimulated with cytokines for 48 h or left untreated were incubated with 200 μl PBS (Thermo Fisher Scientific, Waltham, MA) apically for 2 h which was then collected and pooled (hereafter called apical washes). In parallel, the media on the basolateral side of the cells was also collected. All samples were immediately stored at − 80 °C.

The methods applied here for isolating EVs have been described in detail previously for isolation of EVs from plasma [23] (Fig. 1). In brief, the apical washes and basolateral media samples were centrifuged at 300×g for 10 min at 4 °C to remove debris. Six milliliter sample was then loaded onto iodixanol density cushions (Optiprep, Sigma-Aldrich) and centrifuged for 2 h at 4 °C (SW 41 Ti swinging bucket rotor at 97,000 × g_avg, k-factor: 265.1, Beckman Coulter, Brea, CA). After centrifugation, the interphase between 10 and 30%, consisting of the majority of vesicles, was collected in a total volume of 1 ml. This was then run through size exclusion chromatography consisting of Telos columns (Kinesis, Cambridgeshire, UK) packed with sepharose CL-2B beads (GE Healthcare Life Sciences, Marlborough, MA) and 30 fractions of 0.5 ml were collected. Where applicable, fractions were pooled (fractions 1–6, 7–12, 13–18, 19–24, and 25–30), concentrated by centrifugation for 70 min at 4 °C (TLA 100.3 rotor at 116,000 × g_avg, k-factor: 57.6, Beckman Coulter) and finally the pellets were resuspended in PBS.

Samples were diluted in PBS to have suitable particle concentration for measurement. Measurements were performed with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) with the camera sensitivity set to 80 and the shutter set to 100. Data was analysed using the ZetaView analysis software (version 8.2.30.1) with minimum and maximum size of 5 nm and 5000 nm respectively and minimum brightness of 20.

Samples were lysed with RIPA buffer and sonicated 3 × 5 min with vortexing before protein concentration was determined using microBCA assay kit according to manufacturer’s instructions (Pierce™ microBCA Protein Assay Kit, Thermo Fisher Scientific). For functional studies, protein concentration was determined using Qubit 3.0 Fluorometer (Thermo Fisher Scientific) according to manufacturer’s instructions.

Formvar/carbon-coated copper grids (Ted Pella, Redding, CA) were glow discharged before samples were loaded. Grids and samples were incubated for 15 min, fixed sequentially in 2% paraformaldehyde and 2.5% glutaraldehyde, and contrasted in 2% uranyl acetate. The preparations were examined using LEO 912AB Omega electron microscope (Carl Zeiss NTS, Jena, Germany).

Thirty μl from each SEC fraction or 15 μl from the pooled fractions were separated on 10% polyacrylamide gels and further transferred onto PVDF membranes using a Trans-Blot Turbo Transfer system (Bio-Rad, Hercules, CA). Membranes were blocked using TBS containing 0.01% Tween-20 (TBST) and 5% non-fat dry milk and then incubated with primary antibodies against calnexin (1:1000; clone H-70; Santa Cruz Biotechnology, Dallas, TX) and flotillin-1 (1:1000; clone H-104, Santa Cruz Biotechnology) diluted in TBST containing 0.25% non-fat dry milk over night at 4 °C. Following this, membranes were washed in TBST before incubation with secondary antibodies (donkey anti-rabbit IgG HRP-linked F(ab′)₂ fragment 1:10,000 dilution, or sheep anti-mouse IgG HRP-linked F(ab′)₂ fragment 1:10,000 dilution, GE Healthcare) diluted in TBST containing 0.25% non-fat dry milk. Bands were visualised using ECL Prime Western Blotting Detection (GE Healthcare) on VersaDoc 4000 MP (Bio-Rad).

Proteomic profiling was performed by the Proteomics Core Facility at the Sahlgrenska Academy, University of Gothenburg. EV samples (40 μg/sample) were digested using the filter-aided sample preparation as previously described [23, 24]. Digested peptides were labelled using TMT 10-plex isobaric mass tagging reagents (Thermo Scientific) according to manufacturer’s instructions. Sodium deoxycholate was removed by acidification with trifluoroacetic acid and the sample was further purified using High Protein and Peptide Recovery Detergent Removal Resin (Thermo Fisher Scientific). The TMT-set was fractionated into eight fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific).

Each fraction was analysed on Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) interfaced with nLC 1200 liquid chromatography system. Peptides were trapped on an Acclaim Pepmap 100 C18 trap column (100 μm × 2 cm, particle size 5 μm, Thermo Fisher Scientific) and separated on an in-house constructed analytical column (300 × 0.075 mm I.D.) packed with 3 μm Reprosil-Pur C18-AQ particles (Dr. Maisch, Ammerbuch-Entringen, Germany) using a 75 min gradient from 7 to 80% acetonitrile in 0.2% formic acid. The Orbitrap Fusion Tribrid mass spectrometer was operated in data-dependent MultiNotch MS3 mode. The full scans were acquired at a resolution of 120,000 and the MS2 scans were performed in the ion trap using a collision energy of 35%. The 10 most intense fragment ions were selected for further fragmentation using HCD and a collision energy of 65%. The MS3 scans were acquired at a resolution of 60,000.

RNA isolation and next-generation sequencing was performed as described previously [11] (data from IL-4 + IL-13 stimulated cells is the same as used here). In short, after 24 h stimulation, HBEC-ALI cultures were lysed in QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) and RNA isolated using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions and RNA integrity was assessed (Bioanalyzer 2100, Agilent, Santa Clara, CA). RNA was converted to mRNA libraries using the TruSeq Stranded mRNA kit (Illumina, San Diego, CA) and sequenced using a High Output flow cell on an Illumina NextSeq500 in 2 × 76 cycles. Differential gene expression was assessed with DESeq2 [25] using raw counts as input. Genes were considered significantly differentially expressed if they had a q < 0.05 with Benjamini-Hochberg multiple correction.

For quantitative PCR (qPCR), total RNA isolated from cells as described above was converted into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA). qPCR was run using the comparative Ct method on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher Scientific) using TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific). Gene expression was determined with probes from TaqMan Real-Time PCR Assays (CCL26, NOS2, ANO1, POSTN, SLC26A4, CSF3, PI3 and CCL20, Thermo Fisher Scientific) and normalized against the housekeeping gene ACTB.

Neutrophils were isolated from whole peripheral blood from healthy volunteers using the MACSxpress® Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to protocol and resuspended in media consisting of RPMI 1640 (GE Healthcare) supplemented with 110 μg/ml of sodium pyruvate (Sigma-Aldrich). Media alone, media with EVs or with 5% FBS (used as positive control, as previously described [26]) were added to the lower wells of a 48-Well Micro Chemotaxis Chamber (NeuroProbe, Gaithersburg, MD) on top of which a gelatin-coated 8 μm polycarbonate membrane (NeuroProbe) was placed. After assembly of the chamber, 170,000 neutrophils were added to the upper wells and the chamber was incubated at 37 °C for 3 h. The chamber was then disassembled and number of migrated cells in the lower wells counted using a Bürker chamber with Türk staining solution (Sigma-Aldrich).

Lists of proteins associated with EVs used to create Table 1 were obtained from EVpedia [27] and Vesiclepedia [28]. Proteins related to EV biogenesis and release were collated from relevant reviews [29‐33] and TIBCO Spotfire was used to analyse this data in Supplementary Fig. 2. GO Term Finder [34] and Ingenuity Pathway Analysis (QIAGEN) were used for pathway analysis, Qlucore Omics Explorer (Qlucore, Lund, Sweden) for principal component analysis and GraphPad Prism v.8 (GraphPad Software, San Diego, CA) for performing paired t-tests and mixed-effects analysis unless otherwise stated.

EVs were isolated from apical and basolateral compartments of HBEC-ALI (Fig. 1) and nanoparticle tracking analysis demonstrated that the majority of particles were found in SEC fractions 8–10 from the apical samples (Supplementary Fig. 1a ). Western blot confirmed the presence of the EV marker flotillin-1 and electron microscopy displayed vesicles with size ≤100 nm (Supplementary Fig. 1b-c ) in these fractions. Soluble proteins eluted beyond SEC fraction 16 (Supplementary Fig. 1a ). Only few particles with no SEC peak nor flotillin-1 were detected in the basolateral sample (Supplementary Fig. 1a-b ). Thus, henceforth only apical samples were analyzed.

HBEC-ALI stimulated basolaterally with T2 or T17 cytokines, or media alone, released EVs that eluted in SEC fractions 7–12 which were again containing flotillin-1 but not the endoplasmic reticulum marker calnexin and were of size ≤100 nm (Fig. 2a-d). The F7–12 pools were used for subsequent studies. Stimulation with T2 and T17 cytokines significantly increased the release of EVs as compared to control cells (Fig. 2e). Together, these results demonstrate that primary HBEC-ALI cultures release EVs at their apical side and that this release is increased when the cells are stimulated with T2 or T17 cytokines.

To determine the response to T2 and T17 cytokines, the gene expression in cytokine-stimulated HBEC-ALI cultures was analyzed by RNA sequencing which resulted in 3646 and 5379 differentially expressed genes, respectively. Analysis of the 20 most upregulated genes for each stimulation (Fig. 3a-b, and Supplementary Table 1) revealed several genes previously described in context of T2 or T17 immune responses. Genes induced by T2 stimulation included C-C motif chemokine 26 (CCL26; also called eotaxin-3) [35], inducible nitric oxide synthase (NOS2) [36] and arachidonate 15-lipoxygenase (ALOX15) [37], whereas genes induced by T17 stimulation included granulocyte colony-stimulating factor (CSF3) [10], C-C motif chemokine 20 (CCL20) [38], and the putative IL-17 receptor E-like (IL17REL) [39]. A few genes such as the ion exchanger pendrin (SLC26A4) [40] were however shown to be induced by both T2 and T17 stimulation. The expression of a selection of genes was confirmed by qPCR in additional HBEC donors after stimulation (Fig. 3c).

The increased release of EVs upon cytokine stimulation (Fig. 2e) might be explained by an increased expression of genes encoding proteins involved in EV biogenesis, transport, or release (Supplementary Fig. 2). A total of 61 such genes were found to be differentially expressed upon T2 and/or T17 cytokine stimulation and out of these, 95% were upregulated compared to non-stimulated cells (Supplementary Fig. 2).

Together, these results demonstrate that distinct gene expression signatures reflecting immune responses involved in airway inflammation, as well as proteins involved in EV biogenesis and release, are induced in airway epithelial cells upon T2 and T17 cytokine stimulation.

To further characterize the bronchial epithelium-derived EVs, mass spectrometry was used to profile the proteome of EVs isolated from three donors. In total, 1358 proteins were identified in all samples (Supplementary Table 2). Of 121 top-ranking EV proteins extracted from the EVpedia [27] and Vesiclepedia [28] databases, 101 (83%) were represented in the HBEC-ALI-derived EVs. This included several common EV proteins such as Rab proteins, annexins and tetraspanins (Table 1). Gene ontology analysis focused on cellular compartments showed that over 60% of the proteins were associated with the top term “extracellular exosome” and there was strong association with the term “apical plasma membrane”, confirming that the isolated vesicles were specifically released from the apical side (Fig. 4a). Further analysis, focused on biological processes, revealed association between the epithelium-derived EV proteome and the terms “multivesicular-body assembly”, “endosomal transport” and “viral budding via host ESCRT complex”, which may suggest that at least a part of the isolated EVs have an endosomal pathway-associated biogenesis (Fig. 4b). Interestingly, “cell-to-cell adhesion” and “leukocyte migration” were also among the top-15 terms. Furthermore, top-15 KEGG pathways associated with the EV proteome also showed association to the endosomal pathway (“endocytosis”) and “adherence junction”/“tight junctions” (Fig. 4c). Together, this indicates that EVs of high purity have been isolated and the pathway analysis suggests possible biological roles such as leukocyte migration and cell adhesion.

As T2 and T17 cytokine stimulation altered the HBEC-ALI transcriptome and increased EV release, quantitative proteomics was performed to determine if also the EV proteome was altered. Principle component analysis showed clear separation between EVs isolated from non-stimulated versus stimulated HBEC-ALIs, as well as between EVs isolated from T2 and T17 cytokine stimulated cells (Fig. 5a).

In total 26 and 55 proteins were significantly upregulated after treatment with the T2 and T17 cytokines, respectively (Fig. 5b-e). Under T2 conditions, the most upregulated EV proteins were inducible nitric oxide synthase (NOS2), sushi domain containing 2 (SUSD2) and dipeptidyl peptidase-4 (DPP4). Proteins most upregulated in T17-derived EVs were granulocyte-colony stimulating factor (CSF3), TNF-α induced protein 3-interacting protein 1 (TNIP1) and dual oxidase 2 (DUOX2). None of these proteins were significantly upregulated in EVs in response to the other stimulus, however, some proteins, such as pendrin (SLC26A4) were increased in EVs released under both stimuli. The EVs contained reduced levels of 19 proteins in response to each stimulus (Figs. 5f-g) such as S100A7 and prominin-1 (PROM1) under T2 stimulation, and cilia and flagella associated protein 97 (CFAP97) and dermacidin (DCD) under T17 stimulation. Further, differentially abundant EV-proteins correlate well with expression of the corresponding genes in the stimulated parent epithelial cells (Supplementary Fig. 3a-b).

Ingenuity Pathway Analysis was used to predict upstream regulators of the proteins significantly altered in cytokine-derived EVs. Under T17 conditions, TNF was the only predicted activator, whereas under T2 conditions, both IL-13 and IL-4 had positive activation scores. These findings together confirm that the observed changes in EV proteins are directly related to the stimuli applied.

Ingenuity Pathway Analysis also suggested that pathways related to neutrophil recruitment might be activated by T17-derived EV proteins whereas the opposite was seen for T2-derived EV proteins (Table 2). Implicated EV proteins were for example CSF3, TNFAIP3, TNIP1 and SAA1 that were increased under T17 conditions (Table 2 and Fig. 6a) and NOS2, CD36 and CD44 which were increased under T2 conditions (Table 2 and Fig. 6b).

To test this possible function in vitro, the capacity of HBEC-ALI-derived EVs to drive migration of neutrophils isolated from blood was determined. Although not reaching statistical significance, there was a trend for T17-derived EVs to induce an increased neutrophil migration compared to media alone, whereas the T2-derived EVs, as suggested by the pathway analysis, were unable to do so. Importantly, the migration of neutrophils towards T17-derived EVs was significantly higher than that towards T2-derived EVs (Fig. 6c). The increase in migration induced by T17-derived EVs showed, in similarity with the positive control, less variation among donors compared to the other EV conditions, suggesting a more true induction of neutrophil migration.