Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, results from supplemental oxygen and mechanical ventilation treatment, which can cause barotrauma, volutrauma, and oxygen toxicity [1
]. Infants with BPD exhibit pathophysiological characteristics such as alveolar simplification, arrested lung growth, impaired vascular development, and abnormal pulmonary function [2
]. To prevent pulmonary inflammation caused by mechanical ventilators, systemic corticosteroid treatment has been used and shown to reduce mortality [6
]. There have been several reports regarding neurodevelopmental impairment in BPD patients with hydrocortisone regimens [8
]. In addition to corticosteroids, pharmacological therapies, such as caffeine, diuretics, bronchodilators, and vitamin A, have been used for the prevention and management of BPD. However, it remains unclear how steroids and other medications reduce BPD, due to uncertainties regarding the dosage, timing, and choice of medication.
Stem cell therapy is a promising treatment for the regeneration of damaged lungs. With the common characteristics of self-renewal, clonogenic potential, and multipotency, mesenchymal stem cells (MSCs) are much more likely to be used in clinical applications [11
]. Several experimental trials and clinical applications of MSCs have been attempted to treat incurable diseases [14
]. There are several ongoing clinical trials of MSCs for the treatment and prevention of BPD [15
]. Paracrine factors secreted by MSCs have the ability to suppress the immune response and regulate various immune cell functions [18
]. The immunomodulatory effects of MSCs are communicated via MSC-secreted anti-inflammatory cytokines. However, the precise underlying mechanisms of MSC-mediated immunomodulation have not been fully clarified due to variations in the local microenvironment. Currently, the heterogeneity and variation in donor MSCs are the limitations of stem cell therapy. Therefore, these limitations in MSC immunomodulatory effects can be overcome by determining the appropriate criteria for highly efficient stem cells.
Recently, the treatment of BPD with MSCs has shown that hyperoxia-induced lung injury is ameliorated with alveolar and vascular remodeling [2
]. Moreover, there is no evidence for the replacement of damaged lung tissue by engrafted MSCs. Secretome analysis has shown that the paracrine actions of MSCs are effective in BPD treatment [22
]. Macrophages, derived from blood monocytes, reside in areas of tissue deterioration. Monocytes differentiate into classically activated macrophages (M1) when cells are exposed to microbicidal activity and they have an antigen-presenting function in tissue. Alternatively activated macrophages (M2) are associated with anti-inflammatory and homeostatic functions linked to fibrosis and wound healing. This diverges from the pro-inflammatory and antigen-presenting functions of M1, which induce the inflammatory cytokines, interleukin (IL)-1α, IL-6, IL-8, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. In contrast to M1, M2 induce the anti-inflammatory cytokines, IL-4, IL-10, IL-13, and Arg1 [24
]. Paracrine factors secreted by MSCs facilitate the polarization from inflammatory M1 to anti-inflammatory M2 [27
]. The broad range of potentially therapeutic proteins secreted by MSCs includes angiogenic factors, growth and trophic factors, chemokines, and anti-inflammatory cytokines [28
]. In the inflammatory environment, TNF-inducible gene 6 (TSG-6) or prostaglandin E2 (PGE2), which are related to macrophage polarization, are released by MSCs [30
]. However, these factors are insufficient to explain all of the anti-inflammatory effects and signaling mechanisms of MSCs. Additional proteins are needed to explain the anti-inflammatory reactions and to control macrophage polarization.
Decorin is a small secreted leucin-rich proteoglycan that influences proteins involved in apoptosis, cell proliferation, transcription, chemotherapy resistance, mitosis, and fatty acid metabolism [33
]. Recent research also suggests that decorin-modified MSCs attenuate lung injury through anti-inflammatory and anti-fibrotic activities. Decorin-overexpressing umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) have been shown to evoke a reduction in up-regulation of the following chemokines and inflammatory cytokines: MCP-1, MCP-3, MIP-2, eotaxin, IFN-γ, IL-12, and TNF-α [37
]. CD44 is relatively upregulated in macrophages recruited following tissue injury and is implicated in many chronic inflammatory diseases [38
]. In this study, we determined the criteria for highly efficient stem cells based on their anti-inflammatory effects. After pre-screening the proteins secreted by MSCs, decorin was selected and confirmed as a potential marker. To verify the therapeutic effect of MSCs on BPD, macrophage polarization involving interactions between decorin and CD44 was investigated. We found that the key regulator, decorin, and the underlying mechanisms of macrophage polarization, enhanced the anti-inflammatory effects of MSCs.
In this study, we investigated the therapeutic effects of UCB-MSCs on BPD and the paracrine factors involved in macrophage polarization. CD44 knockdown in NR8383 rat alveolar macrophages reduced macrophage polarization when co-cultured with MSCs. The transition between M1 and M2 is caused by paracrine actions of MSCs and is associated with therapeutic effects. The paracrine actions involved in macrophage polarization were also abolished in vivo in a newborn rat model of hyperoxic lung injury by siRNA-mediated knockdown of decorin, but not by a scrambled control siRNA. Taken together, our results demonstrated that decorin secreted by MSCs is a key modulator of macrophage polarization to regulate anti-inflammatory reactions.
BPD is a severe chronic lung disease in newborns and infants, caused by hyperoxic conditions due to the long-term use of mechanical ventilation for oxygen supplementation. It results in long-term breathing difficulties and significant morbidity. The rationale for the prevention and treatment of BPD is unclear [4
]. Recently, preclinical studies have been performed to assess MSC injection for the treatment of hyperoxia-induced animal models of BPD [41
]. The therapeutic effects of UCB-MSCs in BPD have been confirmed in ongoing clinical trials worldwide [15
]. Previous studies have demonstrated that immunomodulation by MSCs also regulates the functional of immune cells, including monocytes/macrophages, T cells, B cells, and natural killer cells [47
]. Our results showed that MSCs direct the immunological fate of macrophages. LPS-activated macrophages directly co-cultured with MSCs polarized from an M1 phenotype to an M2 phenotype. Anti-inflammatory M2 expressed high levels of CD163, but low levels of the inflammatory marker, CD11b. Secretion of the anti-inflammatory cytokine, IL-10, by rat macrophages increased after co-culture with MSCs. By contrast, the levels of the pro-inflammatory cytokines, IL-8 and IL-6, significantly decreased in LPS-stimulated macrophages after co-culture with MSCs. We tested three MSC lots from different donors to determine the variation in the immunomodulation capacity of MSCs. These results indicated that MSCs drive the polarization of macrophages towards a less inflammatory state and simultaneously enhance the recovery of damaged tissues.
Next, the cognate receptor on macrophages by which MSCs trigger polarization was investigated. We focused our attention on CD44, a surface glycoprotein known as a controller of the macrophage fusion. According to previous studies, CD44 plays an important role in pulmonary innate immunity. Decreased macrophage recruitment and decreased expression of anti-inflammatory cytokines are observed at the site of lung injury in CD44-knockout mice [38
]. CD44 is a principal receptor for hyaluronan (HA) to activate leukocytes and parenchymal cells at sites of inflammation. Moreover, TSG-6, an anti-inflammatory cytokine mainly secreted by MSCs, enhances the interaction of CD44 with HA in inflammatory environments. CD44 triggers fibroblast migration via TGFβ activation, to repair damaged tissue [39
]. In our study, the beneficial effects of MSCs observed in vitro were abolished by CD44 knockdown in macrophages co-cultured with MSCs. CD44-silenced macrophages failed to polarize into the M2 phenotype after MSC treatment. Levels of the pro-inflammatory cytokines, IL-8 and IL-6, in CD44 knockdown macrophages co-cultured with MSCs did not change compared to their levels in LPS-activated macrophages. Thus, the depletion of CD44 may affect macrophage polarization induced by MSCs. Taken together, these results indicate that CD44 on macrophages is a key modulator of MSC-induced polarization.
Decorin is a small leucine-rich proteoglycan that is closely related in structure to biglycan protein, which induces a Toll-like receptor (TLR) 2/4-dependent signaling cascade in macrophages [54
]. M1 recruitment into the kidney has been studied in renal ischemia/reperfusion injury (IRI) in CD44 knockout mice. In this study, the biglycan-CD44 interaction via TLR4 enhanced M1 autophagy, resulting in an increase in the number of M2, followed by a reduction in tubular damage in the kidney. Previous reports have also suggested that decorin induces Peg3, a master regulator of M1 autophagy [56
]. As a co-receptor for biglycan, CD44 is a key regulator of macrophage recruitment [57
]. The role of decorin on macrophages has also been reported related to macrophage proliferation through induction of p27Kip1
]. There have been previous reports regarding the therapeutic effects of decorin. Over-expression of decorin on UCB-MSCs also attenuates acute inflammation after radiation-induced lung injury in mice by regulating inflammation and immune responses [37
]. Moreover, decorin treatment of scarring that occurs after penetrant central nervous system injury, results in regeneration of axons in the spinal cord [59
]. Overexpression of decorin ameliorates diabetic cardiomyopathy by promoting angiogenesis [60
]. Furthermore, bone marrow-derived mesenchymal stem cells infected with decorin-expressing adenovirus promote the recovery of liver function after fibrotic injury [61
]. Following the result of secretome analysis (Figure S4
), the major increase of VEGF, PTX3, TIMP-2, TSP-1, and decorin was demonstrated. The slight increase of TGF-β1 and decrease of EGF were observed. There may be other critical secreted factors that act similar to decorin, and our research did not preclude this possibility.
Previous and ongoing studies support our pre-screening analysis (Figure S4
), which investigated secreted proteins in co-cultures of MSCs with LPS-activated macrophages and found significant increases in decorin levels. Inflammatory environments induce the production of paracrine factors by MSCs to regulate immune cells. The paracrine factors secreted by MSCs are important to screen to enable the identification of highly efficient MSCs for therapeutic applications. In our study, MSCs were isolated from 10 different donors and co-cultured with LPS-activated macrophages to construct inflammatory environments. The levels of decorin varied depending on the MSC lot co-cultured with LPS-activated macrophages. MSCs were divided into two groups: Those expressing high levels of decorin (MSC-H) and those expressing low levels of decorin (MSC-L). Decorin levels on MSCs determine the extent of recovery of damaged lung tissues in BPD. In addition, we silenced decorin in MSCs to confirm its role in MSC-induced macrophage polarization. MSCs and scrambled siRNA-transfected MSCs polarized macrophages into the M2 phenotype, with high levels of CD163 expression and low levels of CD11b expression. CD44 expression levels on macrophages were up-regulated by MSC treatment. However, this effect was no longer present when MSCs were pretreated with decorin siRNA. Decorin silencing suppressed CD44 expression in MSCs. The anti-inflammatory effects of decorin secreted by MSCs were indicated by a decrease in the secretion of the pro-inflammatory cytokines, IL-8 and IL-6, and enhanced secretion of IL-10 by co-cultured macrophages. Macrophages co-cultured with decorin-silenced MSCs showed an increase in IL-10 secretion and a decrease in IL-8 and IL-6 secretion, compared with macrophages co-cultured with MSCs or scrambled siRNA-transfected MSCs. Thus, our results confirmed that decorin secreted by MSCs attenuated inflammation via interaction with CD44 on macrophages.
In a previous study, we showed that MSCs transform the phenotype and functional properties of macrophages in BPD [19
]. In a BPD model, MSCs were shown to trigger the recovery of injured lung tissues. To confirm decorin as a candidate paracrine factor for the selection of the efficient MSCs, we intratracheally injected MSCs with different levels of decorin expression. The MSC H lot secreted the lowest levels of decorin in the high-expressing group, whereas the MSC L lot secreted the highest levels of decorin in the low-expressing group. BPD rats injected with cells from the MSC H lot showed a recovery of lung tissue, with normal alveoli. MLI levels in BPD rats treated with cells from lot MSC L were not significantly different from those in BPD control rats. Interestingly, there was no difference in survival rates between BPD rats injected with cells from lot MSC H and those injected with cells from lot MSC L. However, a significant decrease in CD11b levels and an increase in CD163 levels were observed in BPD rats injected with cells from lot MSC H. In contrast, BPD rats injected with cells from lot MSC L, which secreted low levels of decorin, had high levels of CD11b expression and low levels of CD163 expression. CD44 expression levels on macrophages were high in BPD rats injected with cells from lot MSC H. The anti-inflammatory effect of lot MSC H was confirmed by both a decrease in the levels of the pro-inflammatory cytokines, IL-8 and IL-6, and an increase in IL-10 levels in lung BALF. Although similar survival rates were seen between BPD rats injected with cells from the MSC H lot and those injected with cells from the MSC L lot, the therapeutic effects of MSC L were greater than MSC H, based on the triggering of macrophage polarization.
To confirm the critical role of decorin secreted by MSCs, decorin-silenced MSCs were injected into BPD rats. Injection of decorin-silenced MSCs did not result in full recovery of damaged lung tissues in BPD rats or the suppression of CD11b expression. Moreover, the expression of CD163 was not detected in BPD rats injected with decorin-silenced MSCs. The suppression of CD44 expression seen in BPD rats injected with decorin-silenced MSCs suggested that decorin is a key regulator of macrophage polarization via CD44. A major question that remains unanswered is whether CD44 induces the TLR4-dependent activation of M1 autophagy, leading to M2 macrophage polarization. Therefore, further research regarding M1 autophagy caused by decorin/CD44 interactions is required. Levels of the pro-inflammatory cytokines, IL-8 and IL-6, in lung BALF were increased in BPD rats injected with decorin-silenced MSCs compared to those injected with MSCs or scrambled siRNA-transfected MSCs. In contrast, IL-10 secretion was suppressed in BPD rats injected with decorin-silenced MSCs. Overall, our results suggested that decorin is a key regulator of macrophage polarization by triggering CD44.
In conclusion, the protective effects of MSC therapy against hyperoxia-induced lung injuries are mediated primarily by their anti-inflammatory effects rather than by their regenerating capacity. Thus, specifically triggering decorin-induced macrophage polarization by MSCs may be a novel strategy for the prevention and therapy of BPD. Decorin triggers CD44-dependent macrophage polarization and repair of damaged lung tissues in BPD. Overall, we postulate that the efficacy of stem cell-mediated therapy in inflammatory diseases and tissue damage could be determined by the paracrine factors secreted from MSCs. The anti-inflammatory effects of macrophage polarization triggered by decorin, via CD44, may contribute to the therapeutic efficacy of MSCs.
4. Materials and Methods
4.1. Cell Preparation and Culture Conditions
UCB samples were collected from human umbilical veins isolated within 24 h after neonatal delivery after informed maternal consent. This protocol was approved by the Institutional Review Board of MEDIPOST Co., Ltd. (MP-2015-6-4). UCB-MSCs, separated from mononuclear cells (MNCs) using Ficoll-Paque™ PLUS (GE Healthcare, Uppsala, Sweden), were washed and cultured in minimum essential medium alpha (α-MEM; Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a 5% CO2
incubator. The culture medium was changed once every 2–3 days, as previously described [62
]. The basic characteristics of the MSCs, such as stemness and multi-lineage potential, are shown in Figure S3
The UCB-MSCs used for these experiments were at passage 6 (p6). To clarify the characteristics of MSCs, we determined the proliferation using population doubling level (PD) calculation and senescence on passages 4 and 6. Each passage was cultured for 5 days and analyzed with the trypan blue exclusion method as described previously [64
]. The population doubling level using the following formula: PD = log (total viable cells at harvest/total viable cells at seed)/log2. The senescence of MSCs was detected by senescence-associated β-galactosidase (β-Gal) staining (Sigma-Aldrich, St. Louis, MO, USA) followed by manufacturer’s instructions.
4.2. In Vitro Inflammation Conditions
Rat alveolar macrophage (NR8383 cells), were purchased form ATCC (American Type Culture Collection, Manassas, VA, USA) and were cultured in F-12K medium with 15% FBS. NR8383 cells (1 × 105) were activated with 1 μg/mL LPS derived from Escherichia coli I55:B5 (Sigma-Aldrich, St. Louis, MO, USA). NR8383 cells activated with LPS were used as a positive control for inflammation. LPS-activated NR8383 cells were co-cultured with 1.9 × 104 MSCs for 3 days. To simulate paracrine actions in a clinical application setting, a direct cell-to-cell contact culture system was used. The supernatants were collected from activated NR8383 cells cultured with MSCs. The quantitative measurement of rat IL-6, IL-8, IL-10, and TNFα (all from R&D systems, Minneapolis, MN, USA) and human decorin (Sigma-Aldrich) levels in supernatants were performed using ELISA.
4.3. Small Interfering RNA-Mediated Knockdown of Target Genes
Human decorin siRNAs (100 nM), rat CD44 siRNAs (50 nM), and scrambled siRNAs (50 and 100 nM; Dharmacon, Lafayette, CO, USA) were transfected for 24 h using DharmaFECT reagent, as recommended by the manufacturer. The sequences of primers used for target genes are described listed in Table 1
4.4. Immunofluorescent Staining
NR8383 cells were incubated with rat monoclonal primary antibody (CD11b, 1:100; Abcam, Cambridge, UK) followed by the appropriate secondary antibody (Cy3-conjugated secondary antibody, 1:350; Jackson ImmunoResearch Europe Ltd., Newmarket, UK). Cell surface glycoproteins CD163 (1:50; Santa Cruz Biotechnology, Dallas, TX, USA), CD44 (1:150; Novus Biologicals, Centennial, CO, USA) on fixed NR8383 cells were stained with rat monoclonal antibodies, followed by Alexa Fluor® 488 (1:350, Jackson ImmunoResearch). Injected human MSCs were detected with an anti-human β2 microglobulin (hβ2MG) antibody (1:100, Santa Cruz, Dallas, TX, USA) with an Alexa Fluor® 488-conjugated secondary antibody (1:350, Jackson ImmunoResearch). Before co-culturing with human MSCs, NR8383 cell nuclei were counterstained with Hoechst 33342 to prevent the staining of human nuclei. Fluorescent images were acquired and analyzed using an LSM 800 confocal microscope (Zeiss, Oberkochen, Germany).
4.5. Western Blotting
Macrophages were transfected with a scrambled siRNA or a rat CD44 siRNA for 24 h. Transfected macrophages were lysed with RIPA buffer to extract protein. A total of 20 μg of each protein extract was electrophoresed on a sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel and then transferred to a nitrocellulose membrane. Blocked membranes were incubated with a primary anti-CD44 antibody (Novus Biologicals, Centennial, CO, USA), followed by horseradish peroxidase-conjugated secondary antibodies. Chemiluminescent intensity of immunoblotted bands was visualized using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). The intensity of each band was normalized to β-actin band intensity (Novus Biologicals).
4.6. Flow Cytometry
The characteristics of MSCs (p6) isolated from ten independent donors were analyzed by flow cytometry. MSCs were collected and labeled with fluorescein isothiocyanate (FITC)-conjugated human CD14, CD45, and human leukocyte antigen (HLA)-DR antibodies (BD Biosciences, Franklin Lakes, NJ, USA). Phycoerythrin (PE)-conjugated human CD73, CD166 (BD Biosciences), CD90, and CD105 (Invitrogen, Carlsbad, CA, USA) antibodies were used to measure stem cell surface markers on MSCs. An isotype control was also included. Washed MSCs were fixed with 1% (v/v) paraformaldehyde (Sigma-Aldrich). Stained cells were analyzed by flow cytometry on a MACSQuant instrument (Miltenvi Biotec, Bergisch Gladbach, Germany).
4.7. Cell Differentiation
The multi-lineage differentiation capacity of MSCs (p4 and p6) was determined by analyzing selective differentiation marker expression. The conditions used for osteogenic, chondrogenic, and adipogenic differentiation of MSCs were adopted from previous studies [65
]. To evaluate osteogenesis, cells differentiated into osteoblasts or osteocytes were stained with alkaline phosphatase (Stemgent, Cambridge, MA, USA). Chondrogenic differentiation was assessed by Safranin O staining (Sigma-Aldrich), which detects proteoglycans on cartilage-like cells obtained from pellet culture. Oil red O staining (Sigma-Aldrich) was performed to detect accumulated lipid droplets in differentiated cells.
4.8. Animal Model
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of MEDIPOST Co., Ltd. (MP-LAR-2017-1-2). This study was also performed in accordance with the institutional and National Institutes of Health guidelines for laboratory animal care. Rat pups (10 g) were delivered from timed pregnant Sprague-Dawley rats (Samtako Bio Korea Co. Ltd., Osan, Korea). Two experimental designs were used, as described in Table 2
Within 10 h after birth, rat pups were randomly assigned to following four groups for the first in vivo experiment: Normoxic control group (normal), hyperoxic lung injury group (BPD), hyperoxic lung injury group with MSC H (BPD + MSC H), or MSC L (BPD + MSC L). The differences between the MSC H group and MSC L group were determined by measuring secreted decorin levels. The second experimental design had five groups: (i) Normal, (ii) BPD, and BPD with (iii) MSCs (BPD + MSC), (iv) scrambled siRNA-treated MSCs (BPD + Con siR-MSC), or (v) decorin siRNA-treated MSCs (BPD + Decorin siR-MSC). The control group was maintained under normoxic conditions, whereas the hyperoxic groups were exposed to hyperoxic chambers in which 90% oxygen was maintained from birth to postnatal (P) day 14, as reported previously [41
]. To avoid oxygen toxicity, nursing mother rats were rotated daily between litters maintained under normoxic and hyperoxic conditions. MSCs (1 × 105
, p6) were washed with saline after washing twice with pre-warmed MEM-α without phenol red. After saline washing, MSC suspensions were prepared in saline and injected intratracheally at P5, as described previously [42
]. The survival rates and health conditions of all rat pups were monitored daily. Rat pups were anesthetized by an intraperitoneal injection of pentobarbital (60 mg/kg) at P14. Eleven to fifteen animals were assigned per group.
4.9. In Vivo Transplantation Immunohistochemistry and Morphometry
Whole lung tissues were obtained from sacrificed rat pups and were fixed with 4% paraformaldehyde. Fixed lung tissues were embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H&E). MLI was used to measure the level of alveolarization by dividing the total length of lines drawn across the lung section by the number of intercepts encountered, as reported previously [42
]. Randomly selected sections (>3) per rat and 100 fields per section were assessed. To confirm the transplantation of injected MSCs in lung tissues, an anti-hβ2MG antibody was used and visualized with an Alexa Fluor®
488-conjugated secondary antibody. To detect rat alveolar macrophages, CD11b, CD163, and CD44 were stained with primary antibodies, followed by detection with Alexa Fluor®
488- or Cy3-conjugated secondary antibodies. Nuclei in lung tissues were counterstained with Hoechst 33342. Stained lung tissues were imaged and analyzed by LSM 800 confocal microscopy. The concentrations of rat IL-8, IL-6, and IL-10 in the BALF samples were determined using ELISA, as described previously [41
4.10. Statistical Analysis
All data are presented as means ± standard deviations (SDs) of the values obtained in experiments performed at least in triplicate. Statistical analysis was performed using a one-way analysis of variance, followed by a least-significant difference (LSD) post-hoc test with Prism 6 software (GraphPad, San Diego, CA, USA). A statistically significant difference was reported if p < 0.05.