Mesenchymal Stem Cell Conditioned Medium Promotes Proliferation and Migration of Alveolar Epithelial Cells under Septic Conditions In Vitro via the JNK-P38 Signaling Pathway

Acute respiratory distress syndrome (ARDS) is a major cause of acute respiratory failure with high morbidity and mortality in critically ill patients [1,2]. It is characterized by high-protein pulmonary edema and severe hypoxemia [1,2] and may result from many clinical insults, including sepsis and pneumonia [1]. The pathophysiological basis of ARDS consists of excessive and protracted alveolar inflammation accompanied by damage to the alveolar epithelial barrier [3,4]. Therefore, the efficient regeneration of an intact alveolar epithelium is crucial to restore normal function of the alveolar barrier [5]. Alveolar type II epithelial cells are primarily responsible for re-epithelialization and restoration of the normal alveolar architecture [5]. This process requires spreading, migration and proliferation of alveolar type II cells, which differentiate and replace necrotic or apoptotic alveolar type .I cells [5].

Mesenchymal stem cell (MSC) with proliferation potential and migration ability to injured or inflammatory sites were recently demonstrated to alleviate pathological impairment and promote rehabilitation of alveolar epithelium in several ARDS models [6]. The mechanisms responsible for enhanced the alveolar epithelium repair include the possible trans-differentiation of MSC to form alveolar type II cells [7] and restorative paracrine effects of MSC synthesized growth factors [7]. However, the low levels of MSC engraftment after transplantation may indicate that their beneficial effects are more likely mediated via their secretion of soluble factors than a long-term presence in repaired tissue [8]. Previous reports have demonstrated that MSC conditioned medium (MSC-CM) improved cutaneous wound healing [7,9] and alveolar epithelial cells wound repair [10,11]. However, data supporting the role of MSC-CM in mediation of alveolar epithelial cells under septic conditions is absent and the signaling pathways involved in this repair process remain unknown.

In this study, we investigated the signaling pathways used by MSC-CM in human alveolar epithelial A549 cells and primary human small airway epithelial cells (SAEC) under septic conditions to influence proliferation and migration, emphasizing the potential involvement of mitogen-activated protein kinase (MAPK). We found that MSC-CM activated Jun N-terminal kinase (JNK) and P38 MAPK in alveolar epithelial cells and confirmed that blocking JNK or P38 MAPK resulted in the inhibition of proliferation and migration. These data indicate that MSC promote epithelial cell repair through releasing a repertoire of paracrine factors via activation of JNK and P38 MAPK.

Human umbilical cords (hUCs) were harvested after obtaining signed informed consent forms from three different human donors, according to the Ethical Guidelines for Research Involving Human Subjects or Human Tissue from Chinese People's Liberation Army Hospital. Human umbilical cord Wharton's jelly-derived MSC (hUC-MSC) were cultured and identified as previously described in our lab [12]. Briefly, hUCs were cut into 1 cm segments, and UC arteries, veins and amnion were removed. The gelatinous tissue was excised and minced into 0.5-1 mm³ pieces. Cells were released by treatment with 0.1% collagenase type II (Sigma-Aldrich, St, Louis, Mo, USA) for 16 h at 37°C. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Co., Carlsbad, USA) containing 10% fetal bovine serum (FBS). MSC at passages 3-5 were used in all the experiments.

A demonstration of MSC differentiation ability was tested by culturing under specific culture conditions. For adipogenesis, confluent cells were cultured in MSC culture growth media supplemented with 5 mg/mL of insulin, 50 mM of indomethacin, 1 mM of dexamethasone and 0.5 mM of 3-isobutyl-1-methylxanthine. Medium was changed twice per week for 3 weeks. Cells were fixed with 10% formalin for 20 min and stained with 0.5% Oil Red O (all from Sigma-Aldrich, St, Louis, Mo, USA) in methanol for 20 min. For osteogenic differentiation, confluent cells were cultured in MSC culture growth media supplemented with 0.1 mM of dexamethasone, 10 mM of β-Glycero-phosphate, and 50 mM of ascorbate. Medium was changed twice per week for 3 weeks. Cells were fixed with 10% formalin for 20 min, and stained with Alizarin Red. For chondrogenesis differentiation, confluent cells were cultured in MSC culture growth media supplemented with 75 mM of ascorbate, 0.1 mM of dexamethasone, 1 mM of sodium pyruvate, 0.75 mM of proline and 50 ng/mL of TGF-β3. Medium was changed twice per week for 3 weeks. Cells were fixed with 10% formalin for 20 min and stained with Alcian Blue.

MSC Phenotypes were determined using flow cytometry. MSC were incubated for 30 min at 4°C with HLA-DR-fluorescein isothiocyanate (FITC), CD11a-FITC, CD45-FITC, CD34-phycoerythrin (PE), CD105-PE, CD73-PE and CD90-FITC with isotypematched IgG FITC, IgG-PE and IgG-APC control antibodies (BD Biosciences, USA). Analysis used a FACScan (BD, USA) for at least 10,000 events using CellQuest software (BD, USA).

Conditioned medium was generated as follows: passage 3 MSCs were cultured to 80%-90% confluence in a T150 culture flask (1 × 10⁷ cells), then washed extensively with PBS, and replenished with 15 mL serum-free DMEM (for testing on AEC) and serum-free SABM (basal media for testing on SAEC) for 72 h prior to harvesting the media for further experimentation. Collected media samples were centrifuged at 3,000 rpm for 10 min to remove cell debris, and filtered through a 0.22 µm filter. Then, the media were concentrated by a factor of 50× using centrifugal filter units with 3 kDa cut-off (Millipore, Billerica, MA) following manufacturer's instructions (3 µL per 1 × 10⁵ cells). All the concentrated MSC-CM was kept at -80°C until use.

A549 cells, an alveolar epithelial cell line were immunostained with rabbit anti-pro SP-C primary antibody (Santa Cruz, USA), a specific marker for Alveolar type II epithelial cells, at 1:200 dilution and Cy3/5-conjugated anti-rabbit secondary was used at a 1:100 dilution for visualization (Santa Cruz, CA, USA). DAPI was used for nuclear staining in all immunocytochemistry assays. Images were acquired by a fluorescent microscope (Olympus IX81, Japan). Human primary small airway epithelial cells (SAEC) were purchased from cultured following manufacturer instruction using SAEC (ScienCell, USA) complete growth media. SAECs were harvested and passaged using Subculture Reagent Pack (ScienCell, USA) according to the instructions. Passage 3 to 4 SAECs were used for all experiments.

The proliferation and viability of A549 cells and SAEC cells were measured using a modified 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) [6]. Briefly, cells were cultured in 96-well plates (Corning Corporation, Corning, NY) at an initial density of 5 × 10³ cells per well. At 60% confluence, cells were divided into three groups: control [0.2% foetal bovine serum (FBS) supplemented DMEM 150 µL for AEC] [SABM (basal media) 150 µL for SAEC]; LPS [(0.2% FBS supplemented DMEM 148.5 µL + 1 mg/mL LPS (E. coli 055:B5; Sigma-Aldrich, St.Louis, MO, USA) 1.5 µL for AEC] [SABM (basal media) 148.5 µL + 1 mg/mL LPS 1.5µL for SAEC] and LPS with MSC-CM [(0.2% FBS supplemented DMEM 142.5 µL + 1 mg/mL LPS 1.5 µL + concentrated MSC-CM 6 µL) for AEC] [SABM (basal media) 142.5 µL + 1 mg/mL LPS 1.5 µL + concentrated MSC-CM 6 µL) for SAEC]. In order to determine the best concentration, we first chose three dosages (3 µL, 6 µL and 12µL) of MSC-CM per well (150 µL). For inhibitors, the cells and inhibitors (20 mmol/L of JNK inhibitor SP600125 and P38 inhibitor SB203580) (Cell Signaling Technology Inc, Danvers, MA) were treated just before plating. At the indicated time points (day 1, 3, 5), 10 µL of the MTT (5 g/L) reagent solution was added into each well, followed by incubation of the microplates at 37°C in 5% CO₂ for 4-5 hours. The supernatant was then discarded and 150 µL of analytically pure dimethyl sulfoxide (DMSO) (Amresco, USA) was added. The 96-well plate was vibrated on a micro-vibrator for 15 min and the optical density of each well was measured at 490 nm by an Elx808 Ultra Microplate Reader (Bio-Tek Inc, USA).

The wound healing assay was performed as previously described [13]. Briefly, cells were cultured as confluent monolayers in 6-well plates and a 200-µL pipette tip was used to scratch the monolayer. After wounding, the cell debris was removed by washing with phosphate buffered saline (PBS). Wounded monolayers were then replenished with either 0.2% FBS supplemented DMEM (2 mL) [SABM (basal media) for SAEC] as negative control, 0.2% FBS supplemented DMEM [SABM (basal media) for SAEC] 1.98 mL + 1 mg/mL LPS 20 µL as positive control, or 0.2% FBS supplemented DMEM [SABM (basal media) for SAEC] 1.94 mL + 1 mg/mL LPS 20 µL + concentrated MSC-CM 40 µL with or without presence of inhibitor SB203580 or SP600125 for 24 hours. Wound images were recorded with a digital camera attached to an inverted light microscope (Nikon Eclipse, TS100) at 0 and 24 hours. The average rates of wound closure were calculated from 3 independent experiments. Each treatment was carried out in duplicate, and the experiments were done 3 times.

For the cell migration assay, A549 cells were resuspended in serum-free medium and adjusted to a density of 2.5 × 10⁵ cells/mL. Transwell inserts (6.5-mm diameter and 8-mm pore size; Corning, Inc.) were loaded with the cell suspensions (100 µL), and either 600 µL of 0.2% FBS supplemented DMEM or 594 µL of 0.2% FBS supplemented DMEM + 1 mg/mL LPS 6 µL or 582 µL of 0.2% FBS supplemented DMEM + 1 mg/mL LPS 6 µL+ concentrated MSC-CM 12 µL with or without presence of inhibitor SB203580 or SP600125 was added to the lower chambers. After A549 cells were incubated for 16 h at 37°C, migrated cells were collected from the lower chambers, stained with crystal violet (Beyotime, Haimen, China) and photographed (20 ×). Five samples from each group were selected for quantification and counted. The number of cells that passed through the pore was counted at 200 × magnification in a blind fashion. Each experiment was repeated at least 3 separate times.

Protein samples (30 mg protein) of cell lysates of A549 or SAEC cells were separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The blots were incubated with primary antibody overnight. The primary antibodies used were anti-p-JNK (Thr183/Tyr185), anti-JNK, anti-p-P38, anti-P38, anti-p-ERK, anti-ERK, ATF-2 and c-Jun (1:1000, Cell Signaling Technology, USA) while anti-β-actin was used at 1:3000 (Cell Signaling Technology, USA). All experiments were independently repeated 3 times.

For identification of secretory proteins in MSC-CM, samples were reduced and alkylated with 1 µL of 1M DTT for 1 hour at 45°C and 25 µL of 200 mM iodoacetamide for 30 minutes at room temperature in the dark followed by overnight digestion with sequencing grade trypsin (2 µg per sample, Promega) [10]. Peptides were analyzed by on-line nanoflow liquid chromatography using the EASY-nLC 1000 system (Proxeon Biosystems, Odense, Denmark, now part of Thermo Fisher Scientific) with 16 cm capillary columns of an internal diameter of 75 µm filled with 3µm Reprosil-Pur C18-AQ resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides were eluted with a linear gradient from 3 - 20% buffer B (99.9%acetonitrile in 0.1% formic acid) for 24 min, 30 - 60% B for 12 min and 80-95% B for 8 min at a flow rate of 300 nl/min. The eluate was electro-sprayed into an Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) using a Proxeon nanoelectrospray ion source. The Orbitrap Elite was operated in a HCD top 10 mode essentially as described [14]. Data was searched against current releases of Uniprot Knowledgebase (UniProtKB) and International Protein Index (IPI).

Results are presented as mean ± SD and analyzed using SPSS 17.0 software. Comparisons of multiple groups were made with a Tukey-Kramer post hoc test after analysis of variance (ANOVA). A P value < 0.05 was considered statistically significant.

The majority of MSC cells displayed a spindle-like shape or fibroblast-like shape (Fig. 1A). MSC differentiated into adipogenic, osteogenic, and chondrogenic mesenchymal lineages (Fig. 1B-D). The cell surface antigen phenotype was assessed by flow cytometry. MSC expressed high levels of CD105 (98.93%), CD73 (98.29%) and CD90 (99.83%) and were considered negative for HLA-DR (0.33%), CD11a (0.21%), CD45 (0.34%) and CD34 (2.3%). The human type II alveolar epithelial cell line, A549 was positive for type II AEC marker pro SP-C.

The results reported in Fig. 2A and Fig. 3A showed that concentrated MSC-CM (50 ×) induced a proliferative response in A549 cells (Fig. 2A) and SACE (Fig. 3A) in a dose-dependent manner. Although MSC-CM had the strongest effect on cell proliferation at a dosage of 12 µL compared with 3 µL or 6 µL per well (96-well plate, 150 µL), we used 6 µL concentrated MSC-CM in the subsequent experiments; taking into account that a high dose of MSC-CM may affect cell permeability. As shown in Fig. 2B, MSC-CM significantly promoted the proliferation of A549 cells under septic conditions at day 1, 3 and 5 (P < 0.05). In addition, MSC-CM also significantly promoted the proliferation of SAEC under septic conditions at day 1 and 3 (P < 0.05) (Fig. 3B).

We next investigated the possible effect of MSC-CM on migration of AEC and SAEC by in vitro wound healing and transwell migration assays (only AEC). In the presence of MSC-CM, the wound was more effectively healed over 24 hours as compared to the control group or the LPS group (Fig. 2C, E and Fig. 3C-D). Those findings were corroborated by a transwell assay in which MSC-CM induced a significant increase in the number of A549 cells detected on the bottom side of the membrane after stimulation by MSC-CM for 16 hours (Fig. 2D-F).

We investigated the potential involvement of JNK, P38 and ERK MAPK in MSC-CM stimulated AEC and SAEC under septic conditions. Rapid phosphorylation of JNK and P38 was observed within 30 minutes after MSC-CM stimulation and reached a maximum at 2 hours after MSC-CM stimulation. Although JNK and P38 phosphorylation decreased with time, it remained significantly elevated for 6 hours (Fig. 4A-D and Fig. 5A-D). We did not observe phosphorylation of ERK in any of the time points after MSC-CM stimulation (Fig. 4E, F and Fig. 5E, F).

To further demonstrate that JNK and P38 MAPK pathway involved in the repair process of AEC and SAEC stimulated by MSC-CM, P38 inhibitor SB203580 and JNK inhibitor SP600125 were applied to A549 cells. As shown in Fig. 6A-D and Fig. 7A-D, when AEC and SAEC were pretreated with 20 mmol/L of the inhibitors, the elevated ATF-2 (downstream of JNK and p38) and c-Jun (downstream of JNK) activity that were induced by MSC-CM was decreased. The JNK and P38 inhibitors markedly inhibited MSC-CM induced proliferation of AEC and SAEC under septic conditions (P < 0.05), although they did not completely abolish the effect of the MSC-CM (Fig. 8A-C and Fig. 9A-C). Data from the wound healing and transwell migration assays show that both signaling inhibitors partially reduced cell migration triggered by MSC-CM (Fig. 8D, E).

In total, 720 proteins were detected in the conditioned medium of hUC-MSC isolated from three donors by LC-MS/MS mass spectrometry. The GO enrichment analysis of biological processes, cellular components, and molecular functions showed that the there were 122 those proteins participate in the process of proliferation and migration (Table 1). All the 122 proteins were mapped by using the KEGG pathway database, and MAPK pathway was obtained. Four proteins including TGF-beta receptor type-1, TGF-beta receptor type-2, Ras-related C3 botulinum toxin substrate 1 and Ras-related C3 botulinum toxin substrate 2 were related with JNK/P38 MAPK pathway.

Our results demonstrated that MSC-CM promoted proliferation and migration of AEC and SAEC under septic conditions by activating both JNK and P38. Blockage of JNK or P38 resulted in a significant reduction in cell proliferation and migration, suggesting that MSC-CM exerts its biological activities on AEC and SAEC in a mechanism that requires both the P38 and JNK MAPK pathways.

Mesenchymal stromal cells (MSC) are a small population of multipotent progenitor cells that can be derived from various human tissues such as bone marrow (BM), placenta, adipose tissue (AT), umbilical cord blood and umbilical cord Wharton's jelly (WJ) [15,16]. In vitro studies have already shown that MSC from different origins varied in differentiation potential, proliferation and secretome [15,17]. WJ-MSCs were demonstrated to secrete higher concentrations of chemokines, pro-inflammatory proteins and growth factors compared with AT-MSCs and BM-MSCs [15,18]. WJ-MSCs may be one of the best sources of MSCs for tissue regeneration in future clinical application.

To develop a suitable in vitro model for ARDS, our primary target cell type was A549 cells; however, we replicated equivalent experiments on SAEC to better mimic the biological feature of primary alveolar epithelial cells. We subjected AEC and SAEC in septic conditions. It was stimulated by LPS, which is a glycolipid of the outermost membrane of gram-negative bacteria and can induce many proinflammatory cytokines like IL-β and TNF-α [19,20]. Under septic conditions the proliferation and migration of the AEC and SAEC was impaired, while cell damage was not obvious in normal medium. Moreover, LPS can also impair the permeability and promote the apoptosis of epithelial cells. Similar results were also obtained in another study which demonstrated that inflammatory cytokines showed synergistic cytotoxic effects on A549 cells [21,22].

The damage to LPS induced AEC and SAEC was alleviated by MSC-CM, which increased the number of metabolically active cells with proliferative capacity and also promoted cell migration. This observation has also been noted by others in an in vitro model of pulmonary epithelial wound repair [10]. These effects were because MSC-CM contained high levels of a myriad of proteins that include growth factors, cytokines and extracellular matrix proteins [9,23]. Many of these secretory proteins are biologically active with anti-inflammatory anti-apoptotic, proliferation enhancing and immunomodulatory functions [24,25]. Indeed, several paracrine mediators that can mediate proliferation and migration effects of MSC have been identified, including insulin-like growth factor (IGF-I), interleukin-10, transforming growth factor-β and fibroblast growth factor 2 by our study and other studies [8,26,27,28,29].

Mitogen-activated protein kinases (MAPKs) are a conserved family of enzymes comprising several subclasses, including ERK, JNKs, and p38s [30,31]. Generally, ERK are preferentially activated by mitogens and growth factors, whereas JNK and P38 are predominantly activated by environmental and genotoxic stresses. Despite the apparently simple architecture of MAPK pathway, the three protein kinases are capable of producing exquisitely specific cellular responses [30,31]. Generally, ERK is considered to be involved in the control of cell proliferation [32]. JNK has dual roles in cell proliferation and survival. The functions of JNK as a positive regulator of proliferation and migration have been supported by a number of previous reports [31,33,34,35]. The work by our group has demonstrated activation of JNK could be induced by MSC-CM. One possible reason was that MSC-CM contained some proteins like TGF-beta receptor type-1, TGF-beta receptor type-2, Ras-related C3 botulinum toxin substrate 1 and Ras-related C3 botulinum toxin substrate 2, which could modulate JNK-P38 signalling. In addition, activation of JNK could promote the proliferation and migration of alveolar epithelial cells under septic conditions and this protective effect could be reversed by the JNK inhibitor SP-600125. Because JNK has the ability to phosphorylate JUN, which is a well-established regulator of cell cycle progression and has profound effects on cell proliferation [33]. Importantly, the protective role of KGF in ALI is believed to be mediated in part by activation of JNK [36]. The potential involvement of P38 in mediating proliferation and migration has also been previously studied [31,37,38]. Jiang et al. demonstrated that the p38 MAPK signaling pathway participates in the regulation of vascular smooth muscle cells proliferation by modulating the expression of cell cycle associated proteins [38]. In addition, the role of P38 in cell proliferation and migration was recently reported in HGF-induced human dental papilla cells [31]. The present data confirm previous studies that focused on other cell types. We also examined the downstream transcription factors of P38 and found that the expression of the transcription factor ATF2 was up-regulated after MSC-CM stimulation. In contrast, P38 activation has also been linked to the negative regulation of cell proliferation by several mechanisms, including antagonistic effects on ERK1/2 and JNK/JUN, the down-regulation of cyclins and the up-regulation of cyclin-dependent kinase inhibitors [35,39]. One possible explanation of these findings is that distinct P38 cascades may regulate non-canonical and even opposing functions in different cell lines and under different stimuli [30]. Although ERK has been shown to be related to cell proliferation and migration under some circumstances [40,41] we did not observe any effect on ERK in A549 cells in our studies.

There are some limitations to this study. First, our in vitro model does not adequately represent the in vivo alveolar environment, where alveolar cells interact with multiple other cell types, including fibroblasts, dendritic cells, and inflammatory cells. Second, we primary used the A549 cells as a model of human AEC, but we recognize the limitations of such a cell line, then we replicated most of our experiments with human primary small airway epithelial cells (SAEC). Even so, those two cells may not respond to all biologic stimuli in the same fashion as primary alveolar epithelial cells. However, due to the limited number of primary human alveolar epithelial cells and the difficulty in isolating those cells, many studies of interest could not be performed with these cells.

In conclusion, the current study demonstrated that MSC-CM promoted proliferation and migration of alveolar epithelial cells under septic conditions. This repair process was dependent on the activation of JNK and p38 MAPK. Although we could not completely replicate clinical conditions with this cell based model, our findings may provide new insights into the complex intracellular processes that convey cytoprotection by paracrine factors and may help to better exploit the potential of cell therapy for ARDS.

None declared.

J. Chen and Y. Li contributed equally to this work.