Exosomes from asbestos-exposed cells modulate gene expression in mesothelial cells

Abstract

Asbestos exposure is a determinate cause of many diseases, such as mesothelioma, fibrosis, and lung cancer, and poses a major human health hazard. At this time, there are no identified biomarkers to demarcate asbestos exposure before the presentation of disease and symptoms, and there is only limited understanding of the underlying biology that governs asbestos-induced disease. In our study, we used exosomes, 30–140 nm extracellular vesicles, to gain insight into these knowledge gaps. As inhaled asbestos is first encountered by lung epithelial cells and macrophages, we hypothesize that asbestos-exposed cells secrete exosomes with signature proteomic cargo that can alter the gene expression of mesothelial cells, contributing to disease outcomes like mesothelioma. In the present study using lung epithelial cells (BEAS2B) and macrophages (THP-1), we first show that asbestos exposure causes changes in abundance of some proteins in the exosomes secreted from these cells. Furthermore, exposure of human mesothelial cells (HPM3) to these exosomes resulted in gene expression changes related to epithelial-to-mesenchymal transition and other cancer-related genes. This is the first report to indicate that asbestos-exposed cells secrete exosomes with differentially abundant proteins and that those exosomes have a gene-altering effect on mesothelial cells.—Munson, P., Lam, Y.-W., Dragon, J. MacPherson, M., Shukla, A. Exosomes from asbestos-exposed cells modulate gene expression in mesothelial cells.

Exposure to asbestos is a main causal factor of several human diseases, including malignant mesothelioma (MM), lung fibrosis (asbestosis), and bronchial carcinoma (1). Notably, lung cancer risk is supra-additively increased when an individual both smokes tobacco and is exposed to asbestos (2). The term asbestos (stemming from the Greek term for inextinguishable) refers to a group of hydrated silicate fibers with a length-to-width ratio >3 and is classified as a category 1 carcinogen (3). The wide use of asbestos for industrial purposes across the world, demonstrates its relevance as a human health hazard, now and for years to come (4). Exposure to asbestos occurs primarily through inhalation with fibers first making contact with the upper respiratory tract. Depending on fiber geometry, some asbestos fibers (such as those that are longer and thinner) penetrate deeper into the lung, and tend to have more deleterious biologic effects (5). The initial, and lasting, assault of asbestos occurs on airway epithelial cells and resident macrophages (6, 7), and, because the mechanisms of asbestos-related disease remains unclear, we hypothesize that these epithelial cells and macrophages exposed to asbestos secrete signature factors that contribute to disease development.

To date, there are no studies implicating exosomes in MM pathogenesis and diagnosis. The latency period after initial exposure to developing malignant disease is 15–60 yr, and once diagnosed, MM is fatal within 6–12 mo (8). We believe that identifying a unique protein secretome from asbestos-exposed cells will contribute to the advancement in knowledge needed to diagnose asbestos exposure and possibly aid in future clinical settings. Because of this need for biomarker identification, the purpose of this current study is to evaluate secreted protein signatures from in vitro asbestos exposure models by focusing on proteins found within the subset of extracellular vesicles, known as exosomes.

Exosomes are membrane-bound vesicles in the size range of 30–140 nm and are of endocytic origin (9). These secreted particles have emerged as attractive tools in biomarker identification and as tools for evaluating biologic phenomena. Exosomes are now known to be more than simple waste disposal, but have vital roles in normal physiology and disease states (10). Identification of protein biomarkers using exosomes is gaining significant traction in the field of disease research and has potential for uncovering new modes of diagnoses (11), similar to the discovery of glypican-1 containing exosomes in the identification of pancreatic cancer (12). In addition, exosomes from MM cells and tumors have been quarried for their proteomic signature (13, 14), but this study is the first of its kind to identify exosomal proteins from asbestos-exposed human cells. This is an essential effort, because to have a more thorough understanding of this disease, we must delve into all aspects of asbestos exposure leading to disease development.

The purpose of this research study is to descriptively outline the protein subsets determined from exosome isolates of asbestos-exposed cells, particularly those unique or up-regulated as compared to non–asbestos-exposed controls.

Furthermore, we are very interested in understanding the mechanism by which MM develops. It is not clear whether mesothelial cells transform via direct interaction with asbestos fibers or by secreted factors from other cells interacting with the fibers. Perhaps it is both, but we envisioned that exosomes are also progenitors of disease by sending molecular cargo to mesothelial cells, from asbestos-exposed epithelial cells or macrophages, that may transform nontumorigenic mesothelial cells to becoming more tumorigenic. The novel idea was developed by our lab that exosomes from asbestos-exposed cells lead to or promote disease in an unexposed region (i.e., the pleura or peritoneum).

We hypothesize that asbestos-exposed cells secrete exosomes containing unique protein cargo that may be informative in the biology of asbestos-related disease states and that these exosomes are capable of biologically altering target mesothelial cells to becoming more tumorigenic.

MATERIALS AND METHODS

Cell culture and treatment

Human bronchial epithelial cells (BEAS2B) and a macrophage cell line (THP-1) were purchased from American Type Culture Collection (Manassas, VA, USA) and were grown as reported before (6, 15) in exosome-free fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA).

Cells were downshifted to reducing medium (0.5% exosome-free medium) for overnight and U.S. National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS; Durham NC, USA; https://www.niehs.nih.gov/) reference sample crocidolite asbestos was added to the cells (5 μg/cm²; 72 h) (16). For THP-1 cells, we performed experiments with and without priming cells with the tumor-promoting agent phorbol myristate acetate (PMA) before adding asbestos. Cells were pretreated with 0.5 μM PMA for 3 h (6). In present experiments, we used exosomes from untreated cells as control. Inert particles were not used, as we have shown in our previous publications that they do not have a significant effect on gene expression or other biologic processes (17–19).

After 72 h incubation with asbestos or controls with no asbestos (and other treatments), conditioned cell culture supernatant was removed for exosome isolation.

Exosome isolation from cell culture medium

Exosomes were isolated using ExoQuick-TC precipitation reagent (System BioSciences, Palo Alto, CA, USA), according to the manufacturer’s protocol (20, 21), incorporating a 0.22 μm filtration step after the first centrifugation to ensure a purer yield of exosomes.

Transmission electron microscopy

Formvar/carbon–coated nickel 200 mesh grids were glow discharged for 60 s, and 5 µl of sample was placed on grid and incubated for 1 min. Excess sample was wicked, and grid was touched to 30 µl water drops with wicking performed between each rinse. Grid was touched to 2 sequential 30 µl drops of 2% aqueous uranyl acetate, excess was wicked, and grids were air dried. Grids were imaged under a transmission electron microscope (1400 TEM; Jeol, Tokyo, Japan) for exosomes.

Dynamic light scattering

Dynamic light scattering (DLS) measurements were made on exosome preparations suspended in PBS with the Zetasizer Nano ZSP System (Zen5600; Malvern Instruments, Malvern, United Kingdom), with a 633-nm He-Ne laser as the light source and the Malvern application software.

Nanoparticle tracking analysis

Exosomes number and size were further assessed by nanoparticle tracking analysis (NTA) using the ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and Software ZetaView 8.02.31.

Scanning electron microscopy for detection of asbestos fibers in exosomes

Exosomes from control or asbestos-exposed cells were imaged with a Jeol 6060 Scanning Electron Microscope to check for presence of asbestos fibers within exosomes.

Characterization of exosomes by antibodies

A few isolated exosome samples from the experiment were characterized by anti-CD81. Two aliquots of isolated exosomes from representative groups were characterized by immunoblot analysis for the presence of exosomal marker CD81 (MilliporeSigma, Burlington, MA, USA) and also for absence of calnexin (Novus Biologicals, Littleton, CO, USA) to rule out contaminating endoplasmic reticulum (ER) vesicles.

Exosome uptake by mesothelial cells

Exosomes from epithelial cells and macrophages were labeled with PKH67 dye (MilliporeSigma) according to the manufacturer’s protocol. Labeled exosomes were suspended in PBS and added to target mesothelial cells and imaged on an IX70 Inverted Light Microscope (Olympus, Hauppauge, NY, USA).

Proteomic analysis on exosome samples

Proteins extracted from equal volume of medium (maximum amounts, <100 µg) were run on SDS-PAGE. Equal amounts (100 ng) of gylceraldehyde-3-phosphate dehydrogenase (GAPDH, G5537-100UN; MilliporeSigma) were added to each sample to control for digestion and labeling efficiencies. The proteins were allowed to migrate 3–5 mm into the separating gel, which were stained with Coomassie Brilliant Blue. Single wide bands in which the proteins still had not been separated were excised, destained with 50% acetonitrile (CH₃CN)/50 mM NH₄HCO₃, and subjected to trypsin digestion protocols (22).

Peptide labeling by tandem mass tags

The labeling procedures were performed according to the manufacturer’s protocols (Thermo Fisher Scientific). In brief, the dried peptides from each sample were resuspended in 102.5 µl of triethyl ammonium bicarbonate, and 0.8 mg of tandem mass tag (TMT) reagents dissolved in 41 µl of CH₃CN was added, followed by brief vortexing and incubation for 1.5 h at room temperature. After incubation, 8 µl of 5% hydroxylamine was added to quench the reactions. Twenty-five microliters from each of the reactions (control, PMA, asbestos, PMA and asbestos for THP-1 experiment, or control and asbestos for BEAS2B experiment) was combined and dried down. The THP-1 samples were further purified by ZipTip (MilliporeSigma). All samples were kept at −80°C until mass spectrometry analysis.

Protein identification by liquid chromatography–tandem mass spectrometry

The purified labeled peptides were resuspended in 5 µl of 2.5% CH₃CN and 2.5% formic acid (FA) in water for subsequent liquid chromatography–tandem mass spectrometry (MS/MS)–based peptide identification and quantification. Analyses were performed on the Q-Exactive Mass Spectrometer coupled to an EasynLC (Thermo Fisher Scientific). Samples were loaded onto a 100 μm × 120 mm capillary column packed with Halo C18 (2.7 μm particle size, 90 nm pore size; Michrom Bioresources, Auburn, CA, USA) at a flow rate of 300 nl/min. Peptides were separated with a gradient of 2.5–35% CH₃CN/0.1% FA over 150 min, 35–100% CH₃CN/0.1% FA in 1 min and then 100% CH₃CN/0.1% FA for 8 min, followed by an immediate return to 2.5% CH₃CN/0.1% FA and a hold at 2.5% CH₃CN/0.1% FA. Peptides were introduced into the mass spectrometer via a nanospray ionization source and a laser-pulled ∼3 μm orifice with a spray voltage of 2.0 kV. Mass spectrometry data were acquired in a data-dependent “Top 10” acquisition mode with the lock mass function activated (m/z 371.1012; use lock masses: best; lock mass injection: full MS), in which a survey scan from m/z 350–1600 at 70,000 resolution (AGC target 1e6; max IT 100 ms; profile mode) was followed by 10 higher-energy collisional dissociation MS/MS scans on the most abundant ions at 35,000 resolution (AGC target 1e5; max IT 100 ms; profile mode). MS/MS scans were acquired with an isolation width of 1.2 m/z and a normalized collisional energy of 35%. Dynamic exclusion was enabled (peptide match: preferred; exclude isotopes: on; underfill ratio: 1%; exclusion duration: 30 s). Product ion spectra were searched using the Seaquest and Mascot search engines on Proteome Discoverer 2.2 (Thermo Fisher Scientific) against a curated Human Uniprot (Homo sapiens protein database; 3AUP000005640; http://www.uniprot.org/proteomes/UP000005640). Common processing and consensus workflows for reporter-based quantification were used with minor modifications. In the processing workflow, the following parameters were set as follows: 1) full trypsin enzymatic activity; 2) maximum missed cleavages, 2; 3) minimum peptide length, 6; 4) mass tolerance at 10 ppm for precursor ions and 0.02 Da for fragment ions; 5) dynamic modifications on methionines (+15.9949 Da: oxidation), dynamic TMT6plex modification (The TMT6plex and TMT10plex have the same isobaric mass) on Ntermini and lysines (229.163 Da); and 6) static carbamidomethylation modification on cysteines (+57.021 Da). Percolator node was included in the workflow to limit the false-positive rates to <1% in the data set.

Statistical analysis

In the consensus workflow of Proteome Discoverer 2.2, parameters were set as follows: 1) both unique and razor peptides were used for quantification; 2) Reject Quan Results with Missing Channels: False; 3) Apply Quan Value Corrections: False; 4) Co-Isolation Threshold: 50; 5) Average Reporter S/N Threshold, 10; 6) Total Peptide Amount was used for normalization; and 7) Scaling Mode was set “on All Average.” Ratio calculation was Summed Abundance Based. For hypothesis testing, “background based” ANOVA was used for analyzing the 2 independent experiments of THP-1 cells [2 separate 4plex TMT runs (2 SDS-PAGE), control, treated with asbestos, treated with PMA, or treated with both PMA and asbestos], and the “individual proteins” ANOVA was used for the experiment with BEAS2B cells [2 technical replicates were run for the 6plex TMT with 3 biologic replicates, (control C1-3, asbestos-treated A1-3) incorporated]. P values and adjusted P values (Benjamini-Hochberg method) were calculated accordingly. Only proteins identified in all replicates were kept. For THP1 data, fold changes (asbestos/control; PMA/control; PMA + asbestos/control) from the 2 biologic replicates with CV >20% were eliminated.

All the protein identification and quantification information (<1% FP; with protein grouping enabled) was exported from the Proteome Discoverer result files to Excel spreadsheets for further statistical analyses. The normalized and scaled (to total peptide amount) values were then imported into the JMP Pro 13 (SAS Institute, Cary, NC, USA) to construct the heat maps.

Validation of proteins by immunoblot analysis

Exosomal proteins were validated by immunoblot analysis using antibodies specific to vimentin (Cell Signaling Technology, Danvers, MA, USA), thrombospondin, superoxide dismutase (SOD2), and glypican-1 (Abcam, Cambridge, United Kingdom), as published in Thompson et al. (23). Proteins selected for validation were of biologic relevance to asbestos exposure and cancer.

Exposure of human mesothelial cells to isolated exosomes

Primary human pleural mesothelial (HPM3) cells were purchased from Brigham and Women’s Hospital (Boston, MA, USA) and cultured (18). Exosomes isolated from asbestos-exposed, or unexposed control cells (either BEAS2B or THP-1) were suspended in PBS. For the BEAS2B exosome experiment, either 10 or 20 µg of exosome protein was added to target mesothelial cells every day for 4 d. For the macrophage experiment, equal volumes of exosome preparation rather than protein content from different groups (to take in consideration of different number of exosomes released per different conditions), were added to mesothelial cells. After 96 h of treatment, mesothelial cells were harvested, and total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany).

PCR array to assess the effect of BEAS2B exosomes on epithelial-to-mesenchymal transition–related genes in mesothelial cells

Effect of BEAS2B exosomes on mesothelial cells was analyzed by PCR Array using epithelial-to-mesenchymal transition (EMT) template (Qiagen) to assess the gene expression patterns of genes (23).

Microarray analysis to assess the effects of THP-1 exosomes on mesothelial cells

RNA quality from THP-1 exosome–exposed mesothelial cells was assessed before microarray analysis by using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and subsequently, the RNA was analyzed with the Clariom S Assay (Thermo Fisher Scientific) microarray for human samples. This assay was chosen for a wider breadth of potential gene expression changes outside of the more narrowed view of 1 pathway. Microarray data analysis was performed using the Transcriptome Analysis Console 4.0 (Thermo Fisher Scientific). Our parameters were set to any gene that was expressed differently by both 2- and 1.5-fold up or down at values of P < 0.05.

The NIH Database for Annotation, Visualization and Integrated Discovery (DAVID; https://david.ncifcrf.gov/) was used to classify functional annotation and pathway analysis for genes that were expressed differently in our experimental groups.

Gene expression changes validated by quantitative RT-PCR

Validation of expression changes in selected genes of interest (related to asbestos exposure and/or cancer) was conducted by quantitative RT-PCR after cDNA synthesis from 1 µg RNA by reverse transcription with AMV Reverse Transcriptase (Promega, Madison, WI, USA), according to the manufacturer’s protocol (23). We used Assays on Demand primers and probes for E-cadherin, IL-1ra, desmoplakin, cyclin B2 (CCNB2), early growth response1 (EGR1), Franconi anemia complementation group D2 (FANCD2), ER oxidoreductase-β (ERO1B), cysteine rich with EGF-like domains 2 (CRELD2), and jagged 1 (JAG1) (Thermo Fisher Scientific).

RESULTS

Exosome isolation and characterization from BEAS2B cells

Successful isolation of exosomes was characterized by transmission electron microscopy (TEM), DLS, and Western blot analysis for exosomal marker CD81 (Fig. 1). TEM indicated membrane-bound vesicles in the size range indicative of exosomes, along with the characteristic depressed spherical shape (or “cup shape” as some describe) of exosomes imaged by TEM. DLS showed vesicle populations in the size range of exosomes. Scanning electron microscopy showed no presence of asbestos fibers inside the exosomes. No samples showed any significant signal for calnexin, suggesting no contamination of the ER.

Figure 1.

Exosome isolation and characterization from human bronchial epithelial (BEAS2B) cells and THP-1 macrophages. A) TEM showing exosome membrane-bound structures and proper size range. Scale bars, 100 nm. B) DLS indicating exosome size distribution. C) NTA indicating exosome size distribution and concentration of particles. D) Western blot analysis for presence of exosome marker CD81.

Proteomic analysis of exosomes from asbestos-exposed and control BEAS2B cells showed a different signature

Proteomics profiling was conducted on exosomes from BEAS2B cells, asbestos-exposed or control, using isobaric TMT tags. We identified a total of 145 proteins and compiled a list of proteins with significant differential abundances (55 proteins with P ≤ 0.05, and 34 proteins with adjusted P ≤ 0.05) in the asbestos-exposed group when compared to the control group (Fig. 2A, B and Table 1). A few proteins that increased in abundance in the asbestos group are plasminogen activator inhibitor 1, vimentin, 14-3-3 protein sigma, thrombospondin, transitional ER ATPase, and glypican-1 (Table 1).

Figure 2.

Proteomic analysis of exosomes from asbestos-exposed and control BEAS2B cells showed different protein signatures. A) Heat map indicating the abundances of all proteins identified in both groups. B) Enlarged heat map section showing all differentially abundant proteins with values of P < 0.05 (1 TMT experiment: asbestos vs. control, 3 biologic replicates, 2 technical replicates), listed according to fold change. C) Validation immunoblot of exosomal proteins glypican-1 and thrombospondin.

TABLE 1.

Proteins with significantly increased differential abundance in exosomes collected from asbestos-exposed BEAS2B cells, as compared to exosomes collected from untreated cells

Accession	Description	Fold Change	P	Adj. P
P05121	Plasminogen activator inhibitor 1	1.706	4.90E−04	1.24E−02
P10643	Complement component C7	1.667	1.42E−02	5.22E−02
P07437	Tubulin β chain	1.666	2.68E−02	7.19E−02
P68363	Tubulin α-1B chain	1.585	1.21E−03	1.29E−02
P08670	Vimentin	1.582	1.60E−02	5.67E−02
P01031	Complement C5	1.54	3.29E−03	1.96E−02
P02545	Prelamin-A/C	1.515	2.54E−04	1.24E−02
P05067	Amyloid β A4 protein	1.481	2.69E−03	1.68E−02
P21333	Filamin-A	1.466	3.89E−04	1.24E−02
Q15582	TGF-β-induced protein ig-h3	1.419	5.23E−04	1.24E−02
P06733	Alpha-enolase	1.415	9.23E−04	1.24E−02
P07355	Annexin A2	1.391	3.91E−04	1.24E−02
P31947	14-3-3 protein sigma	1.377	3.92E−02	9.42E−02
Q14767	Latent-TGF-β- binding protein 2	1.371	2.16E−03	1.60E−02
P62979	Ubiquitin-40S ribosomal protein S27a	1.34	9.33E−04	1.24E−02
P62826	GTP-binding nuclear protein Ran	1.334	5.89E−03	2.98E−02
P55072	Transitional endoplasmic reticulum ATPase	1.333	5.64E−03	2.97E−02
P62937	Peptidyl-prolyl cis-trans isomerase A	1.319	1.48E−02	5.36E−02
P01579	IFN-γ	1.315	3.38E−02	8.60E−02
P60709	Actin, cytoplasmic 1	1.313	6.05E−04	1.24E−02
Q16270	IGF-binding protein 7	1.308	7.28E−04	1.24E−02
Q12805	EGF-containing fibulin-like extracellular matrix protein	1.306	2.50E−03	1.68E−02
P00338	L-lactate dehydrogenase A chain	1.306	2.60E−03	1.68E−02
P00736	Complement C1r subcomponent	1.295	5.37E−03	2.87E−02
P07996	Thrombospondin-1	1.292	1.43E−02	5.24E−02
P11142	Heat shock cognate 71 kDa protein	1.285	1.46E−03	1.35E−02
O43707	Alpha-actinin-4	1.285	2.31E−02	6.53E−02
Q00610	Clathrin heavy chain 1	1.274	2.04E−02	6.15E−02
B9A064	Immunoglobulin λ-like polypeptide 5	1.267	2.42E−02	6.74E−02
P0C0L4	Complement C4-A	1.263	7.24E−04	1.24E−02
P14618	Pyruvate kinase PKM	1.263	1.84E−02	5.95E−02
P68431	Histone H3.1	1.259	2.17E−02	6.27E−02
P08758	Annexin A5	1.248	1.27E−02	4.77E−02
Q14624	Interα-trypsin inhibitor heavy chain H4	1.232	9.00E−03	4.13E−02
P04406	GAPDH	1.219	1.60E−03	1.40E−02
Q15149	Plectin	1.215	1.41E−05	1.70E−03
P35579	Myosin-9	1.211	7.55E−03	3.69E−02
P68104	Elongation factor 1-α 1	1.207	1.05E−02	4.38E−02
P39748	Flap endonuclease 1	1.201	1.10E−02	4.51E−02
O94985	Calsyntenin-1	1.19	2.27E−02	6.52E−02
P09871	Complement C1s subcomponent	1.183	1.21E−02	4.77E−02
P02675	Fibrinogen β chain	1.17	5.78E−03	2.98E−02
P04075	Fructose-bisphosphate aldolase A	1.17	1.74E−02	5.78E−02
O43854	EGF-like repeat and discoidin I-like domain- containing protein 3	1.168	1.42E−02	5.22E−02
P35052	Glypican-1	1.163	4.18E−03	2.33E−02
F8WCM5	Insulin, isoform 2	1.138	1.81E−02	5.88E−02
P01024	Complement C3	1.135	1.82E−03	1.52E−02
P21810	Biglycan	1.127	4.04E−03	2.29E−02
Q14980	Nuclear mitotic apparatus protein 1	1.113	1.64E−02	5.67E−02
Q9UBX5	Fibulin-5	1.102	1.90E−02	5.97E−02
P49747	Cartilage oligomeric matrix protein	1.056	1.69E−02	5.72E−02

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as differentially expressed in the asbestos-exposed group with identified tryptic peptides sharing no sequence identity with those from yeast GAPDH, which was added as a control.

Two proteins identified by proteomic analysis were validated by immunoblot analysis

Western blot analysis of glypican-1 and thrombospondin validated that these exosomal proteins are increased from epithelial cells exposed to asbestos (Fig. 2C). Because of the secretory nature of the exosome, no normalization control was included, and equal loading of proteins was verified by Ponceau staining (data not shown).

Exosomes from asbestos-exposed epithelial cells are taken up by mesothelial cells

A set of isolated exosomes were PKH67 labeled and added to mesothelial cells to verify that epithelial cell exosomes interact and are taken up by mesothelial cells. Green fluorescence–labeled exosomes were identified inside mesothelial cells, suggesting their uptake by those cells (Fig. 3A). Monitoring different areas of dish showed a consistent 50–60% cells positive for labeled exosomes.

Figure 3.

Exosomes from asbestos-exposed epithelial cells are taken up by mesothelial cells and caused altered gene expression. A) PKH67 labeled exosomes from BEAS2B cells interact with and were taken up by target mesothelial cells. Scale bar, 100 nm. B) qPCR validation of differentially expressed genes [cadherin 1 (CDH1), desmoplakin (DSP), and interleukin-1 receptor antagonist (IL1RN)] from a PCR array of mesothelial cells after exposure of the cells to exosomes from asbestos-exposed BEAS2B cells (n = 3/group). Results not significant, by 2-tailed Student’s t test.

Exosomes from asbestos-exposed epithelial cells caused altered gene expression in mesothelial cells

To assess whether exosomes from asbestos-exposed epithelial cells are capable of altering the gene expression pattern of mesothelial cells, we repeatedly added isolated exosomes from exposed or control epithelial cells added to mesothelial cells. After 96 h, mesothelial cell RNA was isolated and analyzed by PCR array for EMT. The data indicated multiple gene changes; the top 10 most up- or down-regulated in response to exosomes from asbestos-exposed cells are listed in Table 2. From this list, 3 genes were validated by qRT-PCR (Fig. 3B), E-cadherin, desmoplakin, and IL-1 receptor antagonist (IL-1RN). We observed a downward trend in these 3 genes in response to exposure to exosomes from asbestos-exposed cells, as compared to control exosomes (Fig. 3B).

TABLE 2.

PCR array analysis showing top 10 up- and down-regulated genes in HPM3 cells exposed to asbestos-treated BEAS2B exosomes

Gene name	Fold change	P
Up-regulated
Six transmembrane epithelial antigen of the prostate 1	1.298	1.83E−02
Zinc finger E-box binding homeobox 1	1.2646	7.25E−01
Versican	1.198	1.54E−01
Vacuolar protein sorting 13 homolog A (S. cerevisiae)	1.1452	1.40E−01
Calcium/calmodulin-dependent protein kinase II inhibitor 1	1.1322	2.91E−01
Notch 1	1.1207	6.04E−01
Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1	1.1198	3.16E−01
Tetraspanin 13	1.1198	1.97E−01
PTK2 protein tyrosine kinase 2	1.1171	1.50E−01
Pleckstrin 2	1.1126	1.89E−01
Down-regulated
Matrix metallopeptidase 3 (stromelysin 1, progelatinase)	0.5824	3.05E−01
Fibroblast growth factor binding protein 1	0.6299	6.80E−02
Cadherin 1, type 1, E-cadherin (epithelial)	0.6597	4.86E−02*
Desmocollin 2	0.7248	1.03E−01
IL-1 receptor antagonist	0.7297	3.25E−02*
Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)	0.746	7.46E−02
Desmoplakin	0.7474	8.24E−03*
SRY (sex determining region Y)-box 10	0.7577	9.83E−02
Bone morphogenetic protein 7	0.7594	1.02E−01
Goosecoid homeobox	0.7594	1.02E−01

^*P ≤ 0.05.

Exosomes isolated and characterized from THP-1

Exosomes from THP-1 cells were isolated and characterized for their purity, size, and intactness, by using antibodies, NTA, DSL, and TEM (Fig. 1). DLS and NTA showed vesicle size populations in the size range of exosomes, with some discrepancies between samples analyzed on both machines. This anomaly could be explained by the difference in how each instrument analyzes particles and particle sizes, and perhaps the population of very small (∼10 nm) vesicles may be an artifact of DLS, as this population is not observed with the more robust technique of NTA on the same sample. TEM data shows intact, membrane-bound exosomes in the size range of 40–140 nm (Fig. 1). The presence CD81 and absence of calnexin demonstrated the purity of the preparation (Fig. 1D).

Proteomic analysis showed increased abundance of proteins in asbestos-exposed exosomes from THP-1

Exosomes were isolated from 4 different groups of THP-1 cells; control non–asbestos-exposed; asbestos-exposed; PMA-primed, asbestos-exposed; and PMA-primed, non–asbestos-exposed cells.

Proteomic analysis of TMT-labeled macrophage exosomal proteins from all groups provided a total list of 785 identified exosomal proteins, many of which showing moderate alterations in abundance between groups (Fig. 4A and Table 3). PMA priming had no added effect on exosomal protein signature, compared with the asbestos-alone (no priming) group (data not shown), suggesting that, unlike other stimuli, asbestos exposure to human macrophages do not require priming.

Figure 4.

Proteomic analysis showed proteins of differential abundances in asbestos-exposed exosomes from THP-1. A) Heat map indicating the abundance of all proteins identified in both groups, sorted according to P value (1, 2 indicate 2 separate TMT experiments). B) Expanded heat map region showing exosomal proteins with differential abundances between control group and asbestos-exposed group with P < 0.05 (asbestos vs. control, 2 biologic replicates), listed according to fold change. C) Validation immunoblot of exosomal proteins vimentin and SOD2.

TABLE 3.

Proteins with significantly increased abundance in exosomes collected from THP-1 cells exposed to asbestos compared to control

Accession	Protein name	Fold change	P (I)	Adj. P (I)	P (II)	Adj. P (II)
P39019	40S ribosomal protein S19	1.73	7.00E−11	4.10E−09	1.00E−17	1.70E−14
A0A0C4DFU2	SOD2	1.59	4.40E−16	3.80E−14	1.20E−08	1.40E−06
A0A1W2PQS6	RPS10-NUDT3 readthrough	1.41	3.20E−08	1.70E−06	2.40E−06	1.30E−04
P05387	60S acidic ribosomal protein P2	1.31	1.30E−04	4.70E−03	4.40E−05	1.70E−03
B7Z6Z4	Myosin light polypeptide 6	1.28	3.50E−03	6.50E−02	2.40E−05	1.00E−03
P08670	Vimentin	1.25	1.50E−03	4.30E−02	6.60E−04	1.90E−02
P62277	40S ribosomal protein S13	1.25	4.10E−04	1.40E−02	3.50E−03	7.50E−02
P05386	60S acidic ribosomal protein P1	1.24	4.40E−03	7.70E−02	5.80E−04	1.70E−02
P23141-2	Isoform 2 of liver carboxylesterase 1	1.24	7.50E−05	2.80E−03	2.20E−02	2.40E−01
P62701	40S ribosomal protein S4, X isoform	1.22	7.40E−03	1.20E−01	1.90E−03	4.80E−02
Q14956	Transmembrane glycoprotein NMB	1.22	8.30E−03	1.30E−01	2.30E−03	5.50E−02
P31949	Protein S100-A11	1.2	2.30E−02	2.70E−01	2.10E−03	5.30E−02
J3QRS3	Myosin regulatory light chain 12A	1.19	2.10E−03	4.90E−02	3.30E−02	3.00E−01
P01031	Complement C5	1.18	3.90E−02	3.70E−01	6.50E−03	1.20E−01
P20073	Annexin A7	1.17	1.40E−02	1.90E−01	4.00E−02	3.30E−01

Thirty-two proteins were identified with differential abundance in the asbestos-treated group as compared with the control group (P < 0.05; Fig. 4B and Table 3). Fifteen proteins were in greater abundance from asbestos-exposed groups. The abundance of two of these proteins, SOD2 and vimentin, was validated by Western blot analysis, which found them to be increased in exosomes from asbestos-exposed macrophages (Fig. 4C).

Exosomes from THP-1 cells are taken up by mesothelial cells

A set of isolated exosomes was PKH67 labeled and added to mesothelial cells to verify that THP-1 cell exosomes interact and are taken up by mesothelial cells. Green fluorescence–labeled exosomes were identified inside mesothelial cells suggesting their uptake by the cells (Fig. 5A).

Figure 5.

Exosomes from asbestos-exposed THP-1 cells are taken up by mesothelial cells and caused gene expression changes. A) PKH67 labeled exosomes from THP-1 cells interact with and are taken up by target mesothelial cells. Scale bar, 100 nm. B) Clariom S microarray heat map of gene expression between control mesothelial cells and mesothelial cells exposed to exosomes from asbestos-exposed macrophages. C) Number of differentially expressed genes from microarray analysis in groups of asbestos exosomes vs. control, asbestos fibers vs. control, and asbestos exosomes vs. asbestos fibers. D) Venn diagram showing genes differentially expressed between control mesothelial cells and asbestos exosome exposed cells (A), control- and asbestos fiber–exposed mesothelial cells (B), and the shared genes differentially expressed between both comparisons (A, B). E) qRT-PCR validation of genes up-regulated in asbestos exosome and asbestos fiber groups compared to control (CCNB2, EGR1, and FANCD2) and genes down-regulated in asbestos exosome and asbestos fiber groups compared to control (CRELD2, ERO1B, and JAG1). *P ≤ 0.05, by 1-way ANOVA.

Exosomes from asbestos-exposed THP-1 cells cause gene changes in mesothelial cells

Exosomes isolated from asbestos-exposed and control THP-1 cells were added to mesothelial cells (HPM3). In this experiment, we also included a positive control by exposing a group of mesothelial cells directly to asbestos fibers. After 96 h of exposure, total RNA was extracted and subjected to microarray analysis. Three groups were labeled as follows: control exosomes (0) (cells exposed to no-asbestos THP-1 exosomes), asbestos exosomes (cells exposed to asbestos-exposed THP-1 exosomes), and asbestos fibers (cells directly exposed to asbestos fibers). Cutoff thresholds for analysis were set as anything with a 2-or 1.5-fold transcript-level change with an ANOVA transcript level P < 0.05.

Our main comparison of interest was the asbestos exosome group vs. the control exosome group, while drawing parallels from the asbestos fiber group to the control exosome group. Our results of a 1.5-fold cutoff for the asbestos exosome–exposed mesothelial cells compared with control exosome–exposed mesothelial cells were that 498 genes changed significantly: 241 up- and 257 down-regulated (Fig. 5B, C and Table 4). In addition, the comparison of control exosome–exposed cells to mesothelial cells directly exposed to asbestos fibers yielded differential expressions of 3788 genes: 1803 up- and 1985 down-regulated (Fig. 5C). Of these 2 separate comparisons, there were 206 genes that were mutual in their differential expression profiles (Fig. 5D). With a more stringent cutoff of 2-fold or more up- or down-regulated between asbestos exosomes and control exosomes, we observed 80 significant changes in gene expression levels: 32 up- and 48 down-regulated. A comparison of gene expression profile in mesothelial cells in response to asbestos exosomes and asbestos fibers only is presented in Table 5.

TABLE 4.

Microarray analysis showing top 10 up- and down-regulated genes in HPM3 cells exposed to asbestos administered THP-1 exosomes as compared to control exosomes

Gene name	Fold change	P
Up-regulated
EGR1	3.43	9.60E−03
Meiosis-specific nuclear structural 1	3.22	3.10E−03
Histone cluster 1, H3g	2.82	3.00E−05
Transcript identified by AceView, Entrez Gene ID 4731	2.81	1.00E−03
POTE ankyrin domain family, member C	2.69	3.40E−03
Long intergenic non-protein coding RNA 663	2.54	7.50E−03
PARP1 binding protein	2.53	9.00E−03
Fanconi anemia complementation group D2	2.47	2.40E−02
Transcript identified by AceView, Entrez Gene IDs 79677	2.42	1.48E−02
Chromosome 16 open reading frame 52	2.34	4.02E−02
Down-regulated
CRELD2	0.156	1.30E−03
Glycoprotein Ib (platelet), β polypeptide; septin 5	0.225	1.78E−02
Stromal cell-derived factor 2-like 1	0.271	7.30E−03
ERO1β	0.285	5.00E−04
Schlafen family member 11	0.314	5.00E−03
JAG1	0.321	2.00E−04
Arginase 2	0.344	3.25E−02
Cell division cycle 6	0.351	9.10E−03
Transcript identified by AceView, Entrez Gene IDs 153339	0.365	1.88E−02
GTPase, IMAP family member 2	0.373	1.58E−02

TABLE 5.

Microarray analysis showing top 10 up- and down-regulated genes in HPM3 cells exposed to asbestos administered THP-1 exosomes as compared to asbestos fiber exposure

Gene name	Fold change	P
Up-regulated
Somatomedin B and thrombospondin type 1 domain containing	86.62	3.16E−12
RAB7B, member RAS oncogene family	39.61	3.05E−09
Family with sequence similarity 198, member B	34.44	5.99E−11
Wingless-type MMTV integration site family, member 2B	34.16	4.45E−11
Aldo-keto reductase family 1, member B15	24.6	4.64E−09
SMAD family member 6	23.55	3.04E−09
Aldo-keto reductase family 1, member B10 (aldose reductase)	20.27	2.29E−08
Tetraspanin 8	19.43	2.25E−07
Matrix-remodelling–associated 5	19.4	3.84E−11
Calbindin 2	18.92	1.35E−11
Down-regulated
Lipocalin 2	0.003	1.30E−12
IL-24	0.016	1.43E−12
Endothelial cell-specific molecule 1	0.017	2.31E−12
Paired related homeobox 1	0.018	3.66E−09
Hyaluronan synthase 2	0.018	5.89E−09
Proplatelet basic protein	0.030	1.75E−10
IL-36, β	0.042	3.21E−09
Family with sequence similarity 129, member A	0.049	3.06E−10
Keratin associated protein 21-2	0.051	1.37E−09
Activating transcription factor 3	0.052	5.73E−09

Six common genes of interest were selected from those genes that were differentially expressed upon exposure to either asbestos exosomes or asbestos fibers for validation by qRT-PCR, 3 of which were up- and 3 down-regulated by asbestos exosomes or asbestos fibers upon addition to mesothelial cells, based on known and potential biologic relevance in asbestos exposure. The 3 up-regulated genes were hCCNB2, hEGR1, and hFANCD2, and the 3 down-regulated genes were hCRELD2, hERO1B, and hJAG1. Validation by qRT-PCR showed the same significant trends as in microarray results in all 6 genes (Fig. 5E). CCNB2 was chosen because of its significance in regard to MM, although it was not in our list of top 10 over-expressed genes.

DISCUSSION

Exposure to asbestos fibers is a major human health concern, as it is causally associated with MM, lung cancer, and fibrosis. The scientific and medical communities have yet to delineate a useful set of diagnostic biomarkers for asbestos exposure that may be used to preempt the deadly illnesses that result from inhaling the fibers. Because such inhalation of asbestos fibers is the primary source of exposure and the first cells to contact the fibers are therefore lung epithelial cells and resident macrophages, our study included those cell types.

Our study was conducted to determine exosomal protein abundances in response to asbestos exposure in epithelial cells and macrophages. In addition, as mesothelial cells are specifically susceptible to asbestos, leading to MM, we wanted to gauge the subsequent effect these exosomes may have on mesothelial cells that could be the targets of such exosomes and thereby induce the development of MM. Our rationale is that it is currently unknown whether MM, a tumor arising on the mesothelial cell lining of cavities (i.e., pleura or peritoneum), is the result of direct contact with asbestos fibers migrating from within the lung to the outer lining or of secreted factors (loaded in exosomes) from the original cells to contact the fibers being sent to the mesothelial cells leading to transformation, or perhaps both.

The design herein was to isolate exosomes from asbestos-exposed epithelial cells or macrophages, quarry them for proteomic signatures of asbestos exposure, add the purified exosomes to healthy mesothelial cells, and analyze for gene expression changes that may be involved in MM tumorigenic process.

The study described in this paper is novel, in that we are the first to report on the signature, and potential role, of exosomes in the context of asbestos exposure.

The results of our proteomics analyses of exosomes from asbestos-exposed epithelial cells indicate that a shift in abundance of proteins in epithelial exosomes is caused by asbestos exposure. We observed an increased abundance of thrombospondin-1 in both analyses (MS experiment and immunoblot blot validation) of exosomal proteins from asbestos-exposed epithelial cells. This is interesting given that thrombospondin-1 has been identified as being significantly overexpressed in MM tumors (24). We were also intrigued to see that proteomics analysis indicated higher exosomal abundances of vimentin upon producer cell exposure to asbestos because vimentin is a key regulator in the response to asbestos exposure by regulating the NLRP3 inflammasome and is used as a mesenchymal marker in the transition of mesothelial cells to a more neoplastic state (23, 25). In addition, we validated the increased exosomal abundance of glypican-1 in exosomes from asbestos-exposed cells, which piqued our interest because of its established role as an exosomal indicator of cancer, most notably as a pancreatic cancer biomarker exosome (12, 26).

Next, we studied the effect of epithelial cell exosomes on mesothelial cells. To begin, we confirmed the uptake of said exosomes by using an established method of PKH67 labeling of exosomes and adding to mesothelial cells for visualization of uptake (27, 28). Subsequently, the effect of exosomes from producer epithelial cells (either asbestos-exposed or control) was studied on mesothelial cell transformation genes (EMT pathway). Our rationale for this EMT array was because we have shown recently that asbestos exposure causes mesothelial-to-fibroblastic transition (MFT/EMT) in vitro and in vivo (23). Furthermore, many of the known genetic alterations, either the loss of epithelial-like gene expression or gain of more mesenchymal gene expression, that are found in mesothelial cells and are hallmarks of tumorigenesis and MM occur in EMT genes (29–31).

Our findings were modest changes in multiple EMT genes, consistent with the expectation that exosomes from asbestos-exposed epithelial cells can lead to changes in mesothelial cells similar to those that would occur if the mesothelial cells were in direct contact with asbestos fibers or undergoing transition to a more mesenchymal state. Our PCR array indicated a significant up-regulation in the six-transmembrane epithelial antigen of the prostate-1 gene when mesothelial cells were targeted with asbestos exosomes, and an increased in this gene has been reported as a result of mesothelial cell contact with asbestos fibers (23). We also were encouraged to see that asbestos exosomes lead to marked reduction in IL-1RN, and significant reduction in the expression of the known epithelial markers E-cadherin and desmoplakin. Reduction in E-cadherin and desmoplakin expression are well-described as markers for EMT (32, 33), and these alterations have been described in mesothelial cell exposure to asbestos (34, 35). Our conclusions from these epithelial cell experiments are that there is undoubtedly a signature abundance modification in exosomal proteins from epithelial cells exposed to asbestos fibers and that these exosomes are capable of interacting with, and altering gene signature in mesothelial cells. The lack of significance in some results could be attributable to either shorter duration of exposure of exosomes or a lower concentration of exosomes available to cells.

Next, we studied the macrophages and their influence on mesothelial cells as these are the first cell type to interact with asbestos fibers in the lung along with epithelial cells. Because of the previous reports in the literature that THPs must undergo priming before responding to a stimulus, we performed experiments with and without priming of THPs with PMA before exposure to asbestos. Our proteomic data showed that priming was unnecessary for macrophage exosomes to be affected by asbestos exposure; therefore, those data will not be elaborated upon.

Our results indicate that exposure to asbestos does indeed alter the abundances of certain exosomal proteins in THP-1 cells. We were primarily interested in only those proteins that were increased upon exposure to asbestos, as our quest is to provide data that may lead to biomarker discovery. Those proteins of interest increased in asbestos exosomes from macrophages included vimentin (also shown increased in the epithelial study), SOD2, annexin 5, and we also identified thrombospondin in macrophage exosomes. Vimentin was of interest because of its role in inflammasome initiation and asbestos exposure, as listed above. SOD2 was particularly interesting because of its ability to scavenge oxidants and the fact that it was elevated in asbestos exposure models described elsewhere and in mesothelioma studies (36–39). Our results, followed by validation, indicate that exposure to asbestos leads to differences in protein abundance in exosomes derived from macrophages.

Our next endeavor was to classify whether asbestos exosomes from macrophages have an ability to elicit gene expression changes in mesothelial cells. First, we made sure that exosomes were taken up by mesothelial cells by adding PKH67-labeled exosomes to mesothelial cells and visualizing their uptake.

Microarray data analysis showed that asbestos exosome exposure to mesothelial cells significantly changed the expression of 498 genes compared to cells exposed to control exosomes. Furthermore, as expected and published (18), direct exposure of mesothelial cells to asbestos fibers altered expression of 3788 genes, and of these, 206 genes were commonly differentially expressed by both experimental groups compared with the control. That we see such gene expression similarities indicates that exosomes from asbestos-exposed macrophages undoubtedly have the ability to elicit gene expression changes in mesothelial cells in parallel modes to direct asbestos exposure. Furthermore, finding the gene changes that were not common with direct asbestos exposure is intriguing, as it suggests the capability of exosome contents to affect mesothelial cell gene expression and requires further validation.

To confirm the robustness of our data we validated 6 genes of interest: EGR1, CCNB2, FANCD2, CRELD2, ERO1B, and JAG1. EGR1 is a transcriptional regulator of genes required for cellular differentiation and mitogenesis, has been shown by our group to be increased in cells exposed to asbestos (15), and is involved in mesothelial cell response signaling to asbestos (40). In addition, we were interested in the up-regulated gene CCNB2, a key regulator in cell-cycle machinery, which is involved in TGFβ-meditated cell-cycle control and is involved in the instability of chromosomes, with its overexpression modifying chromosome segregation and the spindle checkpoint (41). Overexpression of CCNB2 is an attribute of MM (42, 43). Also overexpressed in mesothelial cells exposed to asbestos exosomes from macrophages was the regulator of chromosomal stability FANCD2, which is up-regulated in MM caused by asbestos exposure (44, 45).

As for the observed down-regulated mesothelial cell genes, our interest was the ER stress-inducible gene CRELD2 and the oxidoreductase ERO1B involved in ER stress, as asbestos is known to lead to ER stress (46, 47). Last, we noted JAG1, the notch ligand, which is involved in transcriptional regulation in cancer (48).

Our data on asbestos exosomes from macrophages provide robust indication of their ability to mirror many of the gene expression changes of mesothelial cells exposed directly to asbestos fibers. This finding is another addition to the building evidence of the biologic importance of exosomes in disease, and supports our hypothesis that exosomes may be the carrier of information from asbestos-exposed macrophages to mesothelial cells, causing oncogenic changes and MM. Our report is the first to provide insight on the role of exosomes in asbestos-induced mesothelial cell diseases.

This study provides clues to a proteomic signature of exosomes from asbestos-exposed epithelial cells and macrophages that can be further investigated in exosomal biomarker studies in human subjects exposed to asbestos. We are excited to contribute data to the field of exosomes and asbestos research, as there is undeniable evidence that asbestos exosomes are information conduits that alter gene expression in target mesothelial cells. That exosome-induced alteration is remarkably comparable to those changes prompted by direct asbestos fiber contact on mesothelial cells strongly suggests that exosomes are a pivotal player in the human response to asbestos exposure that leads to disease development. There are limitations to this study, including lack of in vivo data and the role of inflammasomes and reactive oxygen species on exosome packaging and secretion. We intend to further develop and validate this study in future in vivo experiments and human serum samples from asbestos-exposed individuals. Further study will confirm and finely delineate the role of exosomes in asbestos exposure biology and provide mines for biomarker discovery.

AUTHOR CONTRIBUTIONS

A. Shukla conceived the study; P. Munson and A. Shukla designed the research; P. Munson and Y.-W. Lam performed the research; P. Munson, Y.-W. Lam, and J. Dragon analyzed the data; P. Munson and A. Shukla wrote the paper; M. MacPherson prepared figures and tables and provided technical help with experiments; and all authors reviewed the manuscript.

Glossary

CH₃CN

acetonitrile

CCNB2

cyclin B2

CRELD2

cysteine rich with EGF-like domains 2

DLS

dynamic light scattering

EGR1

early growth response 1

EMT

epithelial-to-mesenchymal transition

ER

endoplasmic reticulum

ERO1

endoplasmic reticulum oxidoreductase-β

FANCD2

Franconi anemia complementation group D2

JAG1

jagged 1

FA

formic acid

HPM

human pleural mesothelial

MM

malignant mesothelioma

MS/MS

tandem mass spectrometry

NTA

nanoparticle tracking analysis

PMA

phorbol myristate acetate

SOD2

superoxide dismutase

TEM

transmission electron microscopy

TMT

tandem mass tag