Cigarette smoke extract (CSE) was diluted in cell culture media to a final concentration of 0.5%. Recombinant human Wnt5a (rhWnt5a) (Chinese Hamster Ovary Cell Line, CHO-derived Gln38-Lys380) protein was obtained from R&D Systems (Minneapolis, MN, USA) and used at a concentration of 1 µg/ml. PPAR gamma agonist, rosiglitazone (RSG), and antagonist GW9662 were purchased from Sigma-Aldrich (St. Louis, USA) and used at 10 µM concentrations each.

Human blood samples were collected from the Division of Pulmonology, 1st Department of Internal Medicine, Medical School, University of Pecs. A total of 5 COPD patients and 5 age-matched control samples were collected in blood collection tubes (BD Vacutainer, SST: BD SST™ Tubes with Silica Clot Activator and Polymer Gel, Franklin Lakes, NJ, USA). Blood samples were clotted for 30 min at room temperature. Samples were then centrifuged at 1,500 rpm for 10 min. Serum samples were stored at −80°C until used for further analysis.

Mice were anesthetized intraperitoneally with ketamine-xylazine (5 mg/kg), sacrificed by cervical dislocation, and their lungs were excised. Lung tissues were divided into three groups and were stored in RNA-later (Qiagen; Hilden, Germany) for molecular biological studies, in 1 ml lysis buffer for Western blot analysis or in 4% buffered formaldehyde (Szkarabeusz Ltd., Pecs, Hungary) for immunohistochemical examinations.

For the cell sorting procedure, 3 mice/group were anesthetized. Abdominal aorta was intersected, and mice were perfused with 10 ml of PBS through right ventricle to reduce blood content of the lung. To initiate the fine digestion 3 ml trypsin was applied, 10 ml PBS was used to wash out trypsin thereafter. 3 ml collagenase-dispase 3 mg/ml collagenase (Sigma-Aldrich, St. Louis, USA), 1 mg/ml dispase (Roche F. Hoffmann-LaRoche Ltd., Basel, Switzerland), and 1 u/μl DNase I (Sigma-Aldrich, St. Louis, USA) were used to fill up the lungs through the trachea. Pulmonary lobes were dissected into smaller pieces and digested in 10 ml collagenase-dispase for 50 min with continuous stirring. Digested lung cells were filtered with 70 µm cell-strainer (BD Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Single cell suspensions of mouse lungs were labeled with anti-mouse CD45-FITC produced at the University of Pecs, Department of Immunology and Biotechnology and anti-mouse EpCAM1 (G8.8 anti-rat-PE) (40). Cell sorting was performed in a FACSAria III (BD Biosciences, San Jose, CA, USA) cell sorter. The following populations were collected: EpCAM⁻CD45⁻, EpCAM⁺CD45⁻, EpCAM⁺CD45⁺, and EpCAM⁺CD45⁻. The purity of sorting was above 99%.

Cigarette smoke extract solution was prepared using a modification of the method described earlier (41). CS of two 3R4F research grade cigarettes (Kentucky Tobacco Research and Development Center at the University of Kentucky, Lexington, KY, USA) were bubbled into 10 ml of RPMI without FBS (Lonza, Basel, Switzerland) using a Harvard peristaltic pump (Harvard Apparatus, P-1500, Holliston, USA) at a 550 ml/min flow rate for approximately 3 min. CSE solution was filtered through a 0.22 µm filter (TPP, Trasadingen, Switzerland). This solution was considered as 100% stock and diluted with culture media to 0.5% for further experiments. The pH of each CSE solution was measured as a mean pH 7.28. The prepared CSE solution was used within 30 min of preparation. The OD of the inner particles of the CSE was measured spectrophotometrically at the wavelength of 360 nm and compared to the control media.

Human adenocarcinoma A549 cell line was obtained from American Type Culture Collection (Rockville, MD, USA). Transgenic Wnt5a overexpressing-A549 (Wnt5a-A549) cell line was created in our laboratory using lentiviral transgenesis (42). A549 cell lines were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS. Primary human small airway epithelial cells (SAEC), normal human lung fibroblast cells (NHLF), and normal human lung microvascular endothelial cells (HMVEC-L) were cultured according to the manufacturers’ recommendations (Lonza, Basel, Switzerland). Cells were cultured in small airway epithelial cell growth medium (SAGM), fibroblast growth medium, and endothelial growth medium, respectively. All the above cell cultures were incubated at 37°C in humidified atmosphere containing 5% CO₂.

Macrophages were obtained from healthy blood donors (Hungarian National Blood Transfusion Service of Pecs, Pecs, Hungary). Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole peripheral blood by standard density Ficoll-Paque (GE Healthcare, Sigma-Aldrich, St. Louis, USA) gradient centrifugation. Positive selection of monocytes was performed using CD14⁺ MACS colloidal superparamagnetic microbeads (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) conjugated with monoclonal anti-human CD14 antibodies. Cell suspensions were mixed with the beads then CD14⁺ cells were separated by high gradient magnetic separation using MACS MultiStand, OctoMACS (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Monocyte purity was regularly >85%. Macrophage differentiation was induced by incubation with 10 nM PMA, for 5 h, at 37°C in humidified atmosphere containing 5% CO₂.

Three-dimensional lung aggregate tissues were generated as described in previous studies (30, 43, 44). Briefly, NHLF, SAEC, and HMVEC-L all purchased from Lonza (Lonza, Basel, Switzerland) were mixed (40% NHLF, 30% SAEC, and 30% HMVEC-L) in the absence or presence of peripheral monocyte-derived macrophages (30% NHLF, 25% SAEC, 25% HMVEC-L, and 20% macrophages) as described, then the cell suspensions were dispensed onto a low attachment 96-well U-bottom plate (Corning, New York, USA) and were centrifuged at 600 g for 10 min at room temperature. Tissue aggregates were maintained in 2:2:1 ratio of endothelial cell growth medium, SAGM, and fibroblasts growth medium. In the various experiments, aggregates were treated with 0.5% CSE or 1 µg/ml recombinant human Wnt5a (R&D Systems, Minneapolis, USA) for 48 h.

Total RNA was prepared from 3D cell cultures using NucleoSpin RNAII kit (Macherey-Nagel, Düren, Germany) with on-column DNase digestion. Random hexamer primers of the high capacity RNA to cDNA kit (Thermo Fisher Scientific, Waltham, MA, USA) was applied. For cDNA synthesis, 1 µg of total RNA was used according to the manufacturers’ protocol. For gene expression analysis, quantitative RT-PCR was performed using SensiFAST SYBR Green reagent (BioLine, London, UK) on ABI StepOne and StepOnePlus instruments (Thermo Fisher Scientific, Waltham, MA, USA). Data were analyzed using StepOne software and expression changes were normalized to beta-actin housekeeping gene.

Total RNA was isolated from human monocytes then cDNA was synthesized. TaqMan master mix was combined with the cDNA samples then the mixed solutions were loaded onto the Human WNT Pathway, Fast 96-well TaqMan Array (Thermo Fisher Scientific, Waltham, MA, USA) plate. Gene expression was analyzed using ABI StepOnePlus instrument (Thermo Fisher Scientific, Waltham, MA, USA).

Mice were anesthetized with ketamine-xylazine and their lungs were excised. After a formaldehyde fixation of the lung tissue, paraffin embedded sections were made using a microtome. Before proceeding with the staining protocol, the slides had to be deparaffinized by xylene and rehydrated. Antigen retrieval was performed in citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0). Fluorescent staining was performed using rat anti-human Wnt5a (1:100, Clone 442625, R&D Systems, Minneapolis, USA) monoclonal antibody as primary, then Alexa Fluor 488 donkey anti-rat IgG polyclonal antibody (1:200, Thermo Fisher Scientific, Waltham, MA, USA) as secondary antibodies.

The 3D human lung tissue aggregates were carefully removed from the 96-well plate and embedded into Cryomount (Histolab, Finland) embedding medium then immediately frozen to −80^oC. Sections were made using a Leica CM1950 cryostat (Leica, Wetzlar, Germany). After a drying step, sections were fixed in cold acetone (Molar Chemicals Kft., Hungary) for 10 min. Fixed slides were blocked in PBS containing 5% BSA (Sigma Aldrich, St. Louis, USA) for 20 min. Wnt5a protein expression was detected by rat anti-human Wnt5a (1:100, Clone 442625, R&D Systems, Minneapolis, USA) monoclonal antibody as primary antibody. The secondary antibody was Alexa Fluor 488 donkey anti-rat IgG antibody (1:200, Thermo Fisher Scientific, Waltham, USA). Nuclei were counterstained with DAPI (1:1000, Serva, Heidelberg, Germany). Fluorescent images were captured using Olympus IX81 fluorescence microscope (Shinjuku, Tokyo, Japan) equipped with CCD camera and analysis software. Images were processed and analyzed with ImageJ.

Production of pro-inflammatory cytokines IL-6 and IL-8 were measured in cell culture medium using the multiplexed flow CBA Human Inflammatory Cytokines Kit (BD Biosciences, San Jose, CA, USA). For this assay, 50 µl culture medium/each sample of 3D aggregate cell cultures were collected at 48 h time points. The CBA kits were used according to the manufacturer’s instructions. The collected samples were diluted four times in the kit’s assay buffer. Diluted samples (50 µl) were added to equal amounts of fluorescent cytokine capture bead suspension (50 µl), and human inflammatory cytokine-phycoerythrin detection reagent (50 µl). Samples were then incubated for 3 h at room temperature. Labeled beads were measured using FACS Canto II flow cytometer (BD Immunocytometry Systems, Erembodegen, Belgium) with BD FACS DIVA software V6 and data were analyzed by FCS Express V3 software.

Extracellular vesicles were isolated from human serum samples of 5 COPD patients and 5 healthy controls using the Total Exosome Isolation kit (from serum) (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Frozen serum samples were thawed at room temperature. Samples were then centrifuged at 300 × g for 30 min at 4^oC to remove cells and cell debris. Then the supernatant was centrifuged with 2,000 × g for 30 min at 4^oC to collect megasomes/oncosomes in the pellet. The supernatant was then mixed with total exosome isolation reagent and vortexed. Samples were incubated at 2–8°C for 30 min, then centrifuged at 14,000 × g for 10 min at room temperature. The pellets containing EV-s (microvesicles/oncosomes) were collected and resuspended in cold EV resuspension buffer and stored at −80^oC until further analysis.

Extracellular vesicles were isolated from cell culture media following the protocol of the total exosome isolation (from cell culture media) kit (Invitrogen, Cat. number: 4478359). A549 and Wnt5a-A549 cell lines were cultured in DMEM supplemented with 10% FBS at 37°C in humidified atmosphere containing 5% CO₂ until confluent. The medium was then removed, the cell layer washed in PBS, and FBS-free medium was added to the cultures. After 2 days at 37°C, 5% CO₂, the FBS-free culture medium was harvested and centrifuged at 300 × g for 10 min at 4^oC to remove cells and cell debris. Then the supernatant was centrifuged with 2,000 × g for 30 min at 4^oC to collect megasomes/oncosomes in the pellet. Finally, total exosome isolation (for cell culture media) reagent was added in 0.5 volumes. Samples were incubated at 4°C overnight, and then centrifuged at 10,000 × g for 1 h at 4°C. The pellets containing EV-s were collected after centrifugation and resuspended in cold PBS until further analysis. Isolated EV-s were stored at −80°C.

Isolated microvesicles (exosomes) were centrifuged for 1 h at 4°C. Pellets were resuspended in PBS containing DiI (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) (dilution 1:10,000). Samples were incubated for 30 min at 37°C, then centrifuged with 10,000 × g for 1 h at 4°C. Supernatant was then removed and the pellet was resuspended in PBS.

Balb/c mice were injected with equal amount of stained or unstained exosome suspension in 200 µl of PBS. Mice were sacrificed after 20 h of exosome injection. Lung, liver, thymus, and the spleen were removed for fluorescence imaging of extracellular vesicle accumulations in mouse organs by the IVIS Lumina III In Vivo Imaging System using the Living Image Software (IVIS Imaging Systems) (PerkinElmer, Waltham, MA, USA). Fluorescence intensity was compared to fluorescence intensity of unstained control organs.

Microvesicles isolated from cell lines were visualized by electron microscopy. First, microvesicles were resuspended in PBS and then fixed in 2.5% glutaraldehyde aqueous solution. 5 µl of fixed exosomes were embedded in the EM grid, which was flushed by water and blown dry for 24 h. Samples were treated with 5% uranyl-acetate at pH 7 for 5 min. The grid was examined using Morgagni 268D transmission electron microscope at 80 kV. Images were acquired using an integrated MegaView III digital camera (Olympus Soft Imaging Solutions GmbH; Munster, Germany).

Mega EV (oncosome) and micro EV (exosome) samples were resuspended in PBS containing 0.5% Tween 20. 100 µl of each sample was loaded to wells in the Aviva Systems Biology WNT5A ELISA Kit (Human) (OKEH00723). The plate was pre-coated with a Wnt5a-specific antibody (96-well plate, 12 × 8 well strips) and blocked. Standards were also provided by the manufacturer. Samples were incubated for 2 h at 37^oC, then the wells were washed and incubated further with a biotinylated Wnt5a-specific detector antibody for further 1 h at 37^oC. After a washing step, incubation continued with Avidin-HRP conjugate for 1 h at 37°C. The wells were then washed and TMB (3,3′,5,5′-Tetramethylbenzidine) substrate was added for 15 min at 37°C in the dark. The reaction was then stopped and the color reaction was measured at 450 nm absorbance. Results were calculated against the standard curve.

Statistical analysis was performed with GraphPad version 6 software. Data are presented as mean ± SEM, and statistical analysis was performed using the independent samples t-test and one-way ANOVA with Bonferroni correction. p < 0.05 was considered as significant.

To investigate the effects of CS as a systemic inducer of inflammation, in vivo and in vitro studies were performed. First, C57BL/6 mice were exposed to CS for 2 × 30 min each day over 2 months. The test animals developed emphysema-like tissue damage (Figure 1A) that correlated with decreased epithelial cell number (Figure 1B). Epithelial cells were also separated from the CD45⁺ leukocyte population (Figure 1B). Both the epithelial cell enriched and the CD45⁺ cell populations had elevated Wnt5a mRNA levels (Figure 1C). Wnt5a protein expression levels were also increased (Figures 1D,E). Different isoforms of CD45 are expressed in many leukocyte populations including T and B lymphocytes and antigen-presenting cells, no effect of these immune cells, except macrophages, were investigated further. To test whether CSE can trigger Wnt5a up-regulation in human lung tissues, in vitro human lung aggregate cultures were used. The aggregate cultures contained SAEC, mesenchyme (NHLF), and endothelial cells (HMVEC-L) in the presence or absence of human blood monocyte-derived macrophages and were treated with CSE for a maximum of 48 h. The CSE treated aggregates have shown increased levels of Wnt5a both at mRNA and protein levels but only if monocytes were present in the aggregate tissues (Figures 2A–D) (45, 46).

After the recruitment of immune cells to help in eliminating foreign materials and to aid tissue repair after damage or infection, immune reactions are resolved and the tissue homeostasis is normalized. Macrophages are involved in self-limiting, regulated resolution of inflammatory processes at different levels. Tissue-resident macrophages and activated monocytes can adopt different activation status, and although the process is still not fully characterized, M1 and M2 type polarization can provide an outline of how macrophage activity can change during inflammation. To test the effects of CS on the human lung, aggregate cultures of primary human lung cell types were exposed to CSE for 48 h and pro-inflammatory cytokine gene expressions were measured. Both in the presence or absence of macrophages, significant increase of IL6 peptide levels were detected compared to untreated controls (Figure 3A). If the human lung aggregate cultures were exposed to rhWnt5a (1 µg/ml, for 48 h) then both IL-6 and IL-8 peptide levels increased significantly in the monocyte containing tissue cultures indicating a sequential activation of pro-inflammatory cytokine and pro-inflammatory Wnt5a production (Figure 3B). To attempt to dissect such a dynamic process, blood monocytes were exposed to CSE, then, Wnt signaling TaqMan Array was performed (Figures 3C,D). Compared to untreated controls, up-regulation of characteristic non-canonical Wnt signaling molecules were detected, including Wnt5a, Wnt5b, Wnt11 as ligands and an inhibitor of the canonical Wnt pathway, Dickkopf 2 (DKK-2) (Figures 3C,D). In addition to specific molecules of the Wnt pathway, increased transcription of CXXC4—a member of the zinc finger domain-containing protein family—was detected. Up-regulation of CXXC4 that functions as an antagonist of the canonical Wnt/integrated signaling pathway indicates active suppression of the canonical Wnt pathway. Furthermore, increased mRNA levels of CSNKD1 a casein kinase I gene family member, transducin-like enhancer of split 4 (TLE4), and Wnt10a were also detected. While CSNKD1 is an essential serine/threonine-protein kinase and regulator of diverse cellular growth and survival; TLE4 is a transcriptional corepressor. Overexpression of the Wnt10a gene may be of particular interest as Wnt10a can play key roles in carcinogenesis through activation of the Wnt-beta-catenin-TCF signaling pathway. Simultaneously, reduced transcription of Axin2 that plays an important role in the regulation of beta-catenin stability and a characteristic canonical Wnt ligand, Wnt1, were down-regulated. Reduced transcription of TLE2 and secreted frizzled-related protein 5 was also observed, demonstrating deregulation of Wnt signaling in macrophages as a direct effect of CS exposure. Deregulation of either the canonical or the non-canonical Wnt signaling results in modification of PPAR gamma expression and activity (31). To investigate how such changes in the Wnt signaling pathway can affect the dynamic polarization of macrophages, pro-inflammatory cytokines characteristic to the pro-inflammatory M1, and the anti-inflammatory but pro-tumorigenic M2 phenotypes (47) were measured in association with PPAR gamma expression and activity. To study how PPAR gamma is involved in the CS induced Wnt5a up-regulation as well as consequent pro-inflammatory cytokine production, human lung aggregate cultures were exposed to Wnt5a and CSE in the presence or absence of PPAR gamma agonist, rosiglitazone (RSG), and inhibitor (GW9662) (Figures 4A,B). The M1 phenotype is mostly characterized by pro-inflammatory cytokines IL-6, IL-8, IL-23, and IL-1β, while M2 by anti-inflammatory cytokines IL-10 and TGF-beta (34). In the studied time frame, inhibition of the anti-inflammatory PPAR gamma by GW9662 reduced the gene expression of IL-10—the characteristic cytokine marker of the anti-inflammatory M2 phenotype (Figure 4B). Cytokine production, however, is a dynamic process. In order to investigate it, human blood monocyte-derived macrophages were exposed to CSE for two different time points 3 and 48 h, then PPAR gamma transcription was measured. Interestingly, at the 3 h time point the anti-inflammatory PPAR gamma transcription significantly increased, but by 48 h the transcript level reduced to control levels (Figure 4C) indicating that expression of the anti-inflammatory PPAR gamma would not keep up with the prolonged inflammatory stimuli.

The lipo-glycoprotein Wnt ligands are highly hydrophobic (48), therefore, their transport in aqueous body fluids is facilitated by EV-s (49). EV-s (both megasomes/oncosomes or microsomes/exosomes) are now widely accepted as a novel route of cellular communication that can influence the long-range Wnt signal gradients (50, 51). Based on the above, we hypothesized that Wnt5a and low serum levels of pro-inflammatory cytokines can be measured in EV-s isolated from human sera (52, 53). If EV-s can travel from the organ of origin and reach any cells and organs of the human body safely delivering their contents, such transport system would explain the systemic nature of COPD. To test our theory, the larger megavesicles/oncosomes and the smaller microvesicles/exosomes were isolated from sera of COPD patients as well as age-matched healthy controls. The non-small cell carcinoma cell line A549 and its engineered Wnt5a overexpressing sister cell line Wnt5a-A549 were used as positive controls. Wnt5a was measured by ELISA (Figure 5A), whereas pro-inflammatory cytokines were measured by a bead assay using flow cytometry (Figures 5B,C). While Wnt5a was not detectable in sera of neither COPD patients nor age-matched healthy volunteers, megavesicles/oncosomes isolated from sera of COPD patients contained similar levels of Wnt5a as measured in the Wnt5a overexpressing Wnt5a-A549 cell line (Figure 5A). Similarly, while many pro-inflammatory cytokines remained undetectable in pure human sera (data not shown), exosomes contained high levels of such cytokines (Figure 5C) with an extremely high level detected for IL-8 (Figure 5C).

To investigate whether these concentrated “cellular messages” can reach other organs, extracellular vesicles were isolated from the supernatant of the Wnt5a-A549 cell line and the lipid bilayer was stained with a DiI fluorescent dye to be injected into the tail veins of Balb/c mice. Mice were sacrificed after 20 h and the organs were removed to detect fluorescence. The experiments provided evidence that inflammatory cytokine containing microvesicles/exosomes can reach and become concentrated in the liver, lungs, spleen, and thymus of test animals (Figures 5D–F).