Am J Physiol Lung Cell Mol Physiol 315: L87–L101, 2018. First published April 12, 2018; doi:10.1152/ajplung.00505.2017. RESEARCH ARTICLE Activation of the NF-␬B pathway alters the phenotype of MSCs in the tracheal aspirates of preterm infants with severe BPD Tobias Reicherzer,1,2 Susanne Häffner,1,2 Tayyab Shahzad,3 Judith Gronbach,3 Josef Mysliwietz,4 Christoph Hübener,5 Uwe Hasbargen,5 Jan Gertheiss,6 Andreas Schulze,1 Saverio Bellusci,7 X Rory E. Morty,8 Anne Hilgendorff,1,2 and X Harald Ehrhardt1,3 1 Division of Neonatology, University Children’s Hospital, Perinatal Center, Ludwig-Maximilians-University, Campus Grosshadern, Munich, Germany; 2Comprehensive Pneumology Center, Ludwig-Maximilians-University, Asklepios Hospital, and Helmholtz Center Munich, Munich, Germany; 3Department of General Pediatrics and Neonatology, Justus-LiebigUniversity and Universities of Giessen and Marburg Lung Center, Member of the German Lung Research Center (DZL), Giessen, Germany; 4Institute of Molecular Immunology, Helmholtz Center Munich, Munich, Germany; 5Department of Obstetrics and Gynecology, Perinatal Center, University Hospital, Ludwig-Maximilians-University, Munich, Germany; 6 Institute of Applied Stochastics and Operations Research, Research Group Applied Statistics, Clausthal University of Technology, Clausthal-Zellerfeld, Germany; 7Universities of Giessen and Marburg Lung Center, Excellence Cluster CardioPulmonary System, Member of the German Center for Lung Research (DZL), Department of Internal Medicine II, Giessen, Germany; and 8Department of Lung Development and Remodeling, Max Planck Institute for Heart and Lung Research, Member of the German Lung Center (DZL), Bad Nauheim, Germany Submitted 27 November 2017; accepted in final form 29 March 2018 Reicherzer T, Häffner S, Shahzad T, Gronbach J, Mysliwietz J, Hübener C, Hasbargen U, Gertheiss J, Schulze A, Bellusci S, Morty RE, Hilgendorff A, Ehrhardt H. Activation of the NF-␬B pathway alters the phenotype of MSCs in the tracheal aspirates of preterm infants with severe BPD. Am J Physiol Lung Cell Mol Physiol 315: L87–L101, 2018. First published April 12, 2018; doi:10.1152/ ajplung.00505.2017.—Mesenchymal stromal cells (MSCs) are released into the airways of preterm infants following lung injury. These cells display a proinflammatory phenotype and are associated with development of severe bronchopulmonary dysplasia (BPD). We aimed to characterize the functional properties of MSCs obtained from tracheal aspirates of 50 preterm infants who required invasive ventilation. Samples were separated by disease severity. The increased proliferative capacity of MSCs was associated with longer duration of mechanical ventilation and higher severity of BPD. Augmented growth depended on nuclear accumulation of NF␬Bp65 and was accompanied by reduced expression of cytosolic ␣-smooth muscle actin (␣-SMA). The central role of NF-␬B signaling was confirmed by inhibition of I␬B␣ phosphorylation. The combined score of proliferative capacity, accumulation of NF␬Bp65, and expression of ␣-SMA was used to predict the development of severe BPD with an area under the curve (AUC) of 0.847. We mimicked the clinical situation in vitro, and stimulated MSCs with IL-1␤ and TNF-␣. Both cytokines induced similar and persistent changes as was observed in MSCs obtained from preterm infants with severe BPD. RNA interference was employed to investigate the mechanistic link between NF␬Bp65 accumulation and alterations in phenotype. Our data indicate that determining the phenotype of resident pulmonary MSCs represents a promising biomarker-based approach. The persistent alterations in phenotype, observed in MSCs from preterm infants with severe BPD, were induced by the pulmonary inflammatory response. NF␬Bp65 accumulation was identified as a central regulatory mechanism. Future preclinical and clinical studies, aimed to prevent BPD, should focus on phenotype changes in pulmonary MSCs. Address for reprint requests and other correspondence: H. Ehrhardt, Dept. of General Pediatrics and Neonatology, Justus-Liebig-Univ., Feulgenstrasse 12, D-35392 Giessen, Germany (e-mail: harald.ehrhardt@paediat.med.uni-giessen.de). http://www.ajplung.org ␣-SMA; bronchopulmonary dysplasia; mesenchymal stromal cells; NF-␬B; preterm INTRODUCTION Bronchopulmonary dysplasia (BPD) is caused by injury to the developing lung, leading to life-long sequelae (14, 20). Histopathology of BPD shows simplified alveolar structures and dysmorphic capillary configuration (7). The disturbance of lung development and severity of BPD are caused by perinatal and postnatal factors, including prematurity, genetic susceptibility, prenatal and postnatal infections, mechanical ventilation, and oxygen toxicity. These factors cause a pulmonary inflammatory response that is central to the pathogenesis of BPD. BPD is characterized by an imbalance between pro- and anti-inflammatory cytokines, downregulation of vascular and tissue growth factors, influx of inflammatory cells, formation of reactive oxygen species, and activation of proteases (17, 43). The transcription factor NF-␬B is essential for normal lung development, but excessive signaling during pulmonary inflammation is a critical mechanism in abnormal lung development (15, 19, 27, 41). In line with this, the accumulation of proinflammatory cytokines, such as IL-1␤ or TNF-␣, which activate NF-␬B, perturbs normal lung development. Conversely, mechanical ventilation in an oxygen-rich environment can also lead to increased lung damage when NF-␬B signaling and levels of TNF-␣ are reduced (4, 11). Therefore, therapeutic targeting of NF-␬B signaling needs to be critically reevaluated. In recent years, pioneering studies have focused on MSCs obtained from the tracheal aspirates of ventilated preterm infants. These cells fulfilled the classical criteria of MSCs and displayed a lung-specific phenotype, which distinguished them from nonresident MSCs (3, 8, 9, 16, 29, 39). Isolation of MSCs from tracheal aspirates of ventilated preterm infants in these 1040-0605/18 Copyright © 2018 the American Physiological Society Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L87 L88 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA Table 1. Patient characteristics of the study cohort No. of children Gestational age , wk Birth weight, g Male sex Twin birth Early onset infection Antenatal steroids Mechanical ventilatory support, days Any BPD No BPD Mild BPD Moderate BPD Severe BPD Deceased Complete Study Cohort Preterm Infants Fulfilling Inclusion Criteria 112 26 ⫹ 0 (1 ⫹ 4) 786 (241) 70 (62.5%) 46 (41.7%) 63 (56.3%) 103 (92%) 61 (28) 106 (94.6%) 6 (5.4%) 43 (38.4%) 25 (22.3%) 24 (21.4%) 14 (12.5%) 49 25 ⫹ 6 (1 ⫹ 3) 709 (279) 31 (63.3%) 24 (48%) 31 (62%) 46 (92%) 70 (22) 49 (100%) not included 19 (38%) 16 (32%) 14 (28%) not included The relevant characteristics of the entire patient cohort of 112 preterm infants (⬍29 wk of gestational age) and of the subgroup of patients fulfilling study inclusion criteria (see MATERIALS AND METHODS for details) are presented. Children who died during intensive care therapy as a result of sepsis or severe intracranial bleeding before 36 ⫹ 0 wk of gestational age were excluded (n ⫽ 4). The mean values and standard deviations, or the percentage of children, are depicted. Higher order multiples were not present within the study cohort. Early onset infection was diagnosed if the infants showed two typical clinical signs of infection and a pathological immature-to-total neutrophils ratio (I/T) (ⱖ 0.2) and/or an increase in C-reactive protein (CRP) ⱖ 6 mg/l in the first 72 h of life. A positive history of antenatal steroids included the application of a complete course of betamethasone or dexamethasone not longer than 7 days before birth. None of the children within the “no antenatal steroids group” were born beyond 12 h of the initiation of the first course. The parameter “days of mechanical ventilatory support” includes any form of mechanical ventilation or continuous positive airway pressure (CPAP). The severity of bronchopulmonary dysplasia (BPD) was classified according to the definition established by Jobe and Bancalari (21). studies was particularly successful from tracheal aspirates of infants who later developed BPD. This finding led to the conclusion that the release of MSCs into the airway is the result of lung injury. These MSCs demonstrated substantial alterations in the pathways controlled by PDGF receptor-␣, ␤-catenin, and TGF-␤1, which regulate the differentiation of MSCs into myofibroblasts. These alterations were associated with distortion of further septation and interstitial fibrosis (16, 29, 33, 37, 38, 40). We performed detailed descriptive, functional, and molecular studies on MSCs obtained from the tracheal aspirates of preterm infants. We identified a combination of new phenotypic characteristics predictive of a prolonged need for mechanical ventilation and higher severity of BPD. Finally, we mimicked the effects of an inflammatory milieu in vivo by exposing MSCs to proinflammatory cytokines in vitro. This induced phenotype alterations similar to those observed in MSCs isolated from preterm infants with severe BPD. MATERIALS AND METHODS For flow cytometry, the antibodies anti-CD45 (1:50, MHCD4518), anti-CD13 (1:50, MHCD1301), anti-CD105 (1:50, MHCD10505), anti-CD34 (1:50, CD34-581-18), and anti-CD14 (1:50, MHCD1427) were obtained from Caltag (Towcester, UK); anti-CD73 (1:50, 550257), anti-CD90 (1:50, 559869), anti-CD11b (1:50, 557743), and anti-CXCR4 (1:50, 555974) were obtained from BD Biosciences (San Diego, CA). Isotype control antibodies were obtained from BD Biosciences. For Western blot analysis, the antibodies anti-histone H1 (1:500, sc-10806), anti-lamin A/C (1:1,000, sc-6214), and antiNF␬Bp65 (1:500, sc-372) were obtained from Santa Cruz (Santa Cruz, CA); anti-␣-SMA (1:1,000, 113200) was obtained from Calbiochem (San Diego, CA), anti-p-I␬B␣ (1:1,000, 2859) and anti-I␬B␣ (1:1,000, 9242) were obtained from Cell Signaling Technology (Danvers, MA), anti-GAPDH (1:2,500, MA1-22670) and fluorochromeconjugated secondary antibodies were obtained from Thermo Fisher (Waltham, MA). Cytokines were obtained from PeproTech (Hamburg, Germany). All other reagents were obtained from SigmaAldrich (Munich, Germany). All antibodies used in the manuscript can be found in SciCrunch database. Study Cohort, Cell Culture, and Study Parameters A cohort of 112 preterm infants (⬍29 wk of gestational age) from the PROTECT (PROgress in the molecular understanding of The evolution of Chronic lung disease in premature infants Trial) study was eligible for this study. Of these patients, five were excluded because of fungal or bacterial cell culture contamination. No child was excluded because of insufficient sampling. A total of 50 preterm infants met the evaluation criteria of 1) mechanical ventilation for ⱖ7 days and 2) routine suctioning performed at least every other day until successful establishment of MSC cultures. Chorioamnionitis was proven by histopathologic examination. All experiments were approved by the ethics committees of the Ludwig-Maximilians-University Munich (no. 195-07) and the Justus-Liebig-University Gießen (no. 135/12). All MSC samples subjected to cohort analyses were collected at the Munich site. No changes in the ventilation strategies were introduced into the clinical routine during the study period. All procedures involving human subjects were in accordance with the principles of the Helsinki Declaration. Written informed consent was obtained from the parents of all infants. Preservation of Primary Samples, Cell Culture, Transfection Experiments, and Experimental Readouts Cell culture. Cell pellets from tracheal aspirates were resuspended in MesenCult medium supplemented with 20% fetal calf serum (FCS; StemCell Technologies, Vancouver, Canada), 2 mM L-glutamine, 10 mM HEPES buffer solution, 50 U/ml penicillin, 50 ␮g/ml streptomycin, and 50 ␮g/ml gentamicin (Invitrogen, Carlsbad, CA). MSCs were allowed to grow to confluence. Established cultures were maintained under constant growth. The purity of ⬎95% of MSC cultures was determined by cell-surface staining assay described below (3, 16, 33, 39). Experiments were conducted between passages 2 and 6 in DMEM medium (PAN Biotech, Aidenbach, Germany) without FCS. The area of the well, covered by cells at the start of experiments, ranged between 10 and 25%. Fig. 1. Characterization of mesenchymal stromal cells (MSCs). Isolated cells displayed a homogeneous and stable MSC phenotype. A: description of the experimental procedure. MSCs were detected 1⫺4 days after cultivation of tracheal aspirates. MSCs were allowed to grow to confluence within 10⫺16 days before passaging. Experimental procedures were performed between passages 2 and 6. B: using flow cytometry, cells expressed the surface receptors CD13, CD73, CD90, and CD 105 which are typically expressed on MSCs, while they were not expressing markers of hematopoetic or myelopoetic cells (CD11b, CD14, CD34, CD45, and CXCR4). C: cell differentiation into adipogenic (top), osteogenic (middle), and myofibroblastic (bottom) cells was confirmed using Oil Red O staining, alizarin red staining, or immunofluorescence labeling for ␣-smooth muscle actin (␣-SMA). D: stability of cell characteristics was ensured until passage 6 for the proliferation index in n ⫽ 20 different MSC cultures (top) and for the content of ␣-SMA (bottom). Statistical analysis was performed using an ANOVA and Bonferroni post hoc adjusted pairwise comparison. NS, not significant. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L89 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA Cell transfection. Transient transfection was performed with Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. siRNA against NF␬Bp65 (5=-GCCCUAUCCCUUUACGUCA-3= (MWG Biotech, Ebersberg, Germany) and AllStars nega- A tive control siRNA (Qiagen, Hilden, Germany) was used at a concentration of 20 nM. Experiments were started 24 h after transfection. I␬␬2 inhibitor IV (Merck KGaA) was used to inhibit the phosphorylation of I␬B␣. passage 2-6 C flow cytometry passage 1 passage 0 cell death proliferation assays protein analysis 100µm 100µm transfection control day 4 adipocyte differentiation day 10-16 B 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 100 101 102 103 0 100 101 104 102 103 104 100 100 80 80 80 60 60 60 40 40 40 20 20 20 102 0 100 101 103 104 102 103 104 100 100 80 80 80 60 60 60 40 40 40 20 20 20 102 0 100 101 103 104 CD45-PerCP-Cy5.5 103 104 0 100 101 control 102 myofibroblast differentiation 103 104 102 103 104 CD11b-PECy7 CXCR4-PE 1.5 D NS normalized α-SMA expression level 3 proliferation index 102 osteoblast differentiation 104 CD34-PerCP-Cy5.5 100 0 100 101 103 100µm 0 100 101 CD105-PE CD90-APC 102 2 1 0 #50 (severe BPD) 1 0.5 0 2 3 4 5 passage 100µm CD14-FITC 100 0 100 101 control 0 100 101 CD73-PE CD13-FITC 100µm 6 7 3 6 12 15 passage AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L90 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA mild BPD moderate BPD severe BPD 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 47 48 49 51 52 53 54 55 57 58 60 61 used four different antibody panels: one containing CD13-FITC, CD73-PE, CD34-PerCP-Cy5.5, and CD14-APC-Cy7; one containing CD105-PE, CD45-PerCP-Cy5.5, CD14-APC-Cy7, and CD90-APC; one with CD45-PerCP-Cy5.5, CD90-APC, and CD14-APC-Cy7; and one containing CD95-FITC, CXCR4-PE, and CD11b-PECy7. Propidium iodide (1 ␮g/ml) was added to each panel to label and sort out dead cells. Negative controls were stained with an isotype control panel. Flow cytometry was performed using an LSR II device. Facs Diva software version 6.1.3 (BD Biosciences) was used for data acquisition, and FlowJo analysis-software version 8.8.6 (Tree Star, Ashland, VA) was used for analyses. Compensation was performed with leftover cells and compensation beads (BD Biosciences). Cell proliferation assays. For quantification of cell proliferation, cells were plated in a 96-well plate, with density defined as 10 –25% of the well area covered. The change in the well area covered was observed over time using a Cellscreen device and data acquisition using PA adhesion software (Innovatis, Bielefeld, Germany). Manual cell counts were performed in a Neubauer chamber after the addition of trypan blue. Western Blot Analysis Cytosolic extracts were obtained by cell lysis in 10 mM 4-(2hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES, pH 7.0), 1 mM KCl, 1.5 mM MgCl2, and 0.5% Triton-X supplemented with a proteinase inhibitor cocktail I (Merck KGaA). Nuclear extracts were obtained after lysis of cell nuclei in 20 mM HEPES (pH 7.9), 400 mM KCl, 0.1 mM EDTA, and 25% glycerin. Protein density was quantified using AIDA imaging software version 2.50 (Raytest, Straubenhardt, Germany). An internal standard deposited on each gel enabled the comparison between different gels. Histological Staining and Immunofluorescence 0 7 14 21 days Fig. 2. Emergence and duration of the presence of mesenchymal stromal cells (MSCs) in tracheal aspirates. The time point of first detection of MSCs and the duration of successful cultivation did not differ between groups defined by disease severity. Tracheal aspirates were cultured every other day during the entire period of invasive mechanical ventilation. MSC outgrowth is indicated by bars. Flow cytometry. The induction of apoptosis was determined using Nicoletti staining (32). For multicolor flow cytometry, cells were washed in a buffer containing 2% glucose, 1% BSA, 0.1% EDTA, and 0.1% sodium azide. Cells were resuspended and incubated with fluorochrome-conjugated antibodies at a concentration of 1:50. We Cells were incubated for 9⫺18 days in a medium containing dexamethasone (10 ␮mol), isobutylmethylxanthine (100 ␮g/ml), indomethacin (50 ␮mol), and insulin (10 ␮g/ml, Sanofi-Aventis, Frankfurt, Germany) for adipocyte differentiation, and in a medium containing dexamethasone (0.1 ␮mol), ␤-glycerophosphate (10 mmol), and L-ascorbic acid (50 ␮g/ml) for osteoblast differentiation. Culture medium was exchanged every third day. For myofibroblast differentiation, cells were incubated with 1 ng/ml TGF-␤ added to the medium for 48 h (16, 36). Histological detection of adipocytic and osteoblastic differentiation was conducted with Oil Red O or Alizarin Red staining, respectively. Immunofluorescence was performed using sterilized glass slides. Cells were fixed either in methanol or acetone, permeabilized with Table 2. Detection of mesenchymal stromal cells in tracheal aspirates: separation by BPD severity scores BPD Grade No. of infants Gestational age, wk Birth weight, g Chorioamnionitis Day of first MSC isolation Duration of presence of MSCs, days Maximum peak inspiratory pressure until culture establishment Maximum FIO2 until culture establishment, % Mechanical ventilatory support, days Proliferation index Mild Moderate Severe P Value 19 25 ⫹ 3 (7 ⫹ 3) 704 (145) 9/19 (47.4%) 9 (5) 8 (7) 14 (6) 29 (7) 65 (13) 2.04 (0.55) 13 25 ⫹ 1 (0 ⫹ 6) 707 (137) 2/13 (15.4%) 8 (4) 12 (8) 13 (2) 33 (3) 78 (13) 2.7 (0.69) 17 25 ⫹ 3 (1 ⫹ 5) 709 (165) 4/14 (28.6%) 8 (4) 12 (7) 16 (4) 40 (18) 96 (33) 2.87 (1.0) ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬍0.05 ⬍0.05 Tracheal aspirates from 49 preterm infants were cultured at least every 2nd day during the period of mechanical ventilation and screened for the presence of mesenchymal stromal cells (MSCs). The mean values and SDs are presented. The number of patients with proven chorioamnionitis on pathological examination is presented in relation to the total number of patients. No pathological examination could be performed on the placenta of 3 patients with severe bronchopulmonary dysplasia (BPD) (2 home births and 1 outborn child, where placenta was not sent for workup). Statistical analysis was performed using ANOVA on ranks and ␹2 to analyze for the presence of chorioamnionitis. FIO2, fraction of oxygen in the breathing air. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L91 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA Table 3. Detection of MSCs in tracheal aspirates: separation by time point of first appearance First Appearance Within No. of infants Day of first MSC isolation Gestational age, wk Birth weight, g Mild BPD Moderate BPD Severe BPD Maximum peak inspiratory pressure until culture establishment Maximum FIO2 until culture establishment, % Mechanical ventilatory support , days Proliferation index Days 0–7 Days 8–21 P Value 20 5 (2) 24 ⫹ 6 (7 ⫹ 2) 630 (98) 7 (35%) 5 (25%) 8 (40%) 14 (3) 34 (17) 83 (33) 2.6 (0.13) 20 11 (4) 26 ⫹ 0 (7 ⫹ 2) 746 (143) 8 (40%) 4 (20%) 8 (40%) 16 (3) 34 (7) 79 (15) 2.24 (0.77) ⬍0.05 ⬍0.05 ⬍0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 Preterm infants were separated by the time point of the first appearance of mesenchymal stromal cells (MSCs). Parameters were analyzed as in Tables 1 and 2. Statistical analysis was performed using Student’s t-test. BPD, bronchopulmonary dysplasia. C A D 5 start of experiment (0h) 100 * end of experiment (96 h) * 3 2 1 B 0 3 proliferation index #22 (severe BPD) 40 20 0 0 24 #20 severe BPD #21 severe BPD 48 96 incubation time (hours) #22 (severe BPD) E number of dead cells (%) 60 2 1 0 0 24 48 96 incubation time (hours) 60 40 20 #16 (mild BPD) #16 (mild BPD) well area covered (%) 80 cell count (x102) proliferation index 4 0 #20 severe BPD #21 severe BPD 100 75 50 25 NS 0 #20 severe BPD #21 severe BPD Fig. 3. Determination of proliferative capacity of mesenchymal stromal cells (MSCs) and induction of cell death. Cell proliferation was determined using Cellscreen automated computer-based light microscopy. Results were confirmed by complementary techniques. A: the proliferative capacity of MSCs was determined over time using Cellscreen. Red lines indicate the surface area covered by the cells, while blue lines indicate the uncovered surface area excluded from the red marked area. B: the proliferation index was introduced to standardize the well area covered at the start of experiments (right) as cell density varied between samples (left). Cellscreen analysis (C) and manual cell counts (D) yielded identical results in MSCs from different patients. E: the fraction of dead cells did not differ between samples. The mean from n ⫽ 3 independent measurements is shown. Statistical analysis was performed using Student’s t-test. *P ⬍ 0.05, which indicates statistically significant differences. NS, not significant. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L92 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA Triton-X, rinsed in phosphate-buffered saline (PBS), and stained with specific primary antibodies and fluorochrome-conjugated secondary antibodies. Cells were then mounted on slides, and the nuclei were counterstained using Vectashield mounting medium with DAPI (Vector, Burlingame, CA). Images were acquired using a Zeiss Axiovert 200 M fluorescent microscope (Zeiss, Jena, Germany) and OpenLab software version 3.0.8 (Improvision, Coventry, UK). Determination of Cytokine Levels in Tracheal Aspirates Protein expression of IL-1␤ was measured in tracheal aspirates using the IL-1␤ Quantikine ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Standardization to sIgA (Immunodiagnostik, Bensheim, Germany) was performed to compensate for the dilution effects of the suctioning procedures (11). Statistical Analysis The proliferation index (PI) was calculated as the quotient of (well area covered at the end of the experiment)/(well area covered at the start of the experiment). Student’s t-test was used to test for statistically significant differences between two independent groups. Multivariate analyses were performed using an analysis of variance (ANOVA) test, and Bonferroni correction was used to adjust for multiple comparisons. Association studies were analyzed with Spearman’s rank order correlation coefficient, and regression analyses were performed with a standard linear logistic model or a proportional odds model, depending on the type of the response variable (metric/binary/ordinal). We used a linear mixed model with random intercept to test for effects on batches of * A proliferation index 5 4 3 C 2 days mechanical ventilatory support 250 1 0 mild moderate BPD B 1.0 severe proportional-odds-model; β=1.14 p=0.008 estimated probability 0.8 0.6 severe BPD moderate BPD mild BPD 0.4 p=0.025 200 150 100 R2 linear=0.105 50 0 0 1 2 3 4 5 6 proliferation index 0.2 0 1 2 3 4 5 proliferation index Fig. 4. The proliferative capacity of mesenchymal stromal cells (MSCs) as a predictor of the duration of mechanical ventilation and severity of bronchopulmonary dysplasia (BPD). The proliferation index was significantly increased in MSCs from preterms with severe BPD in the cohort from Table 1. A: statistical analysis was performed from 3 independent experiments performed between p2 and p6 using one-way ANOVA and post hoc pairwise comparisons by means of t-tests with Bonferroni adjustment. *P ⬍ 0.05 indicates statistically significant differences. B: the predictive value was verified using a proportional odds model. P ⫽ 0.008 indicates statistically significant differences. C: the association between the PI and days of mechanical ventilatory support was demonstrated using linear regression. P ⫽ 0.025 indicates statistically significant differences. The association remained statistically significant when the two outlying values were omitted from the analysis. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA MSC cultures obtained from different children. A child-specific random intercept was included to account for dependencies between observations of the same child. Differences were considered statistically significant at P values ⬍ 0.05. L93 derived from children with better and poorer pulmonary outcomes. Proliferative Capacity of MSCs As a Predictor of the Duration of Mechanical Ventilation and Severity of BPD RESULTS We performed an observational study of a prospective cohort of 112 preterm infants (⬍29 wk of gestation), and determined the phenotype of MSCs isolated from the tracheal aspirates of these infants. Patient characteristics are described in Table 1. Presence and Characterization of MSCs MSCs were detected in the tracheal aspirates of every preterm infant ventilated for at least the first 7 days of life (data not shown). Cells grew to confluence within 8⫺16 days. Standardized protocols (depicted in Fig. 1A) were started at passage 2. Flow cytometric analyses confirmed the specific phenotype of MSCs and the high purity of cultured cells (9, 16, 33). MSCs were identified by flow cytometry. MSCs were positive for MSC surface markers CD13, CD73, CD90, and CD105 and negative for CD11b, CD14, CD34, CD45, and CXCR4; CD11b, CD14, CD34, CD45, and CXCR4 are markers of hematopoietic precursors, leukocytes, macrophages, dendritic cells, fibrocytes, and endothelial cells and are not expressed on MSCs (Fig. 1B) (9, 16, 26, 31, 34, 44). The characteristic pluripotency of MSCs was confirmed by adipocytic, osteoblastic, and myofibroblastic differentiation (Fig. 1C). Stability of cell characteristics was assured until passage 6 by testing the relevant phenotypic parameters (Fig. 1D). Because 49 of the 50 infants fulfilled the criteria for having BPD, we focused on the degrees of BPD severity (Table 1) (21). Neither the day of first appearance nor the duration of successful cultivation from tracheal aspirates was predictive for the severity of BPD (Fig. 2 and Table 2). As expected, children with high severity of BPD needed prolonged ventilatory support. There was no difference in the distribution of BPD severity between preterm infants with MSC present in tracheal aspirates within the first 7 days of life and those with MSC present in tracheal aspirates only after day 7 and before day 21 of life (Table 3). These data are in agreement with previous observations showing that the presence of MSCs is associated with the development of BPD (3, 16, 39). Therefore, we then evaluated characteristics that could be used to discriminate among MSCs MSCs were grouped according to disease severity into mild, moderate, and severe BPD. MSCs from the three groups did not differ in the density of surface receptor expression and potential for adipogenic, osteogenic, or myofibroblastic differentiation (data not shown). The duration for establishing a successful MSC culture at passage 0 varied highly among the cells obtained from different patients. This observation was reproduced under standardized conditions in cell culture. Automated repetitive light microscopy was used to determine the changes in well area covered over time (Fig. 3A). The proliferation index (PI) was introduced to compensate for differences in the well area covered at the start of the experimental procedures (Fig. 3B). Automated repetitive light microscopy (Fig. 3C) and manual cell counting (Fig. 3D) yielded identical results in selected experiments and indicated that the increase in the well area covered resulted from an increase in absolute cell numbers. Using Nicoletti staining, we ruled out the notion that the difference in absolute cell numbers was a consequence of variations in cell death (Fig. 3E). When MSC samples were separated by disease severity, statistically significant differences in the PI were observed between the groups (Fig. 4A). Using a proportional odds model and logistic regression, followed by inspection of the receiver operating characteristic (ROC) curve, the PI was predictive of BPD severity (Fig. 4B and data not shown). In agreement with this result, a higher PI was associated with longer duration of ventilatory support. The PI was not impacted by early or late time points of first establishing the MSC culture (Fig. 4C and Tables 2 and 3). Thus the severity of BPD can be predicted from alterations in the proliferative capacity of MSCs. Proliferative Capacity of MSCs is Correlated with Accumulation of NF␬Bp65 and Downregulation of ␣-SMA The earliest changes, observed in the lungs of preterm infants who later developed BPD, included the influx of inflammatory cells and an imbalance of inflammatory cytokines and growth factors. Because NF-␬B is a central regulator of most inflammatory processes and proliferation (15, 41), we focused on the contribution of NF-␬B to heterogeneous growth characteristics. MSC samples that displayed a particularly low Fig. 5. The proliferative capacity of mesenchymal stromal cells (MSCs) is correlated with the nuclear accumulation of NF␬Bp65 and reduction in ␣-smooth muscle actin (␣-SMA) expression. Nuclear accumulation of NF␬Bp65 was increased in MSCs with a higher PI. A: nuclear NF␬Bp65 was increased in MSCs (left) that were selected for their high spontaneous proliferation (right). Lamin A served as loading control. The order of samples from the identical blot was rearranged (middle and bottom blot) and indicated by separating lines without any further manipulation. Student’s t-test was used to test for differences between groups; P ⬍ 0.001 indicates statistically significant differences. B: computer-based image quantification was introduced to compare protein density between different gels as presented for nuclear NF␬Bp65. An internal standard deposited on each gel enabled comparison of different gels (standardized expression level). The relative NF␬Bp65 expression was calculated as NF␬Bp65 quantification/Lamin A quantification; standardized quantification was calculated by division by the internal standard. Different sections from identical gels (indicated by separated lines) are presented without any further manipulation. C: the expression level of NF␬Bp65 was significantly higher in preterms with severe BPD when the technique described in B was applied to evaluate the total cohort. Nuclear extracts were available from n ⫽ 42 patients. Statistical analysis was performed using one-way ANOVA and post hoc pairwise comparisons by means of t-tests with Bonferroni adjustment. *P ⬍ 0.05 indicates statistically significant differences. D: the predictive accuracy of NF␬Bp65 was verified using the proportional odds model. P ⫽ 0.015 indicates statistically significant differences. E: inhibition of I␬B␣ phosphorylation by I␬␬2 inhibitor IV (10 ␮M) reduced proliferation in MSCs after 72 h. Western blot analysis was performed after 48 h. Statistical analysis was performed using a post hoc Bonferroni adjusted pairwise comparison. The mean from n ⫽ 3 experiments is shown. *P ⬍ 0.005 indicates statistically significant differences. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L94 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA or high PI were selected and assayed for nuclear accumulation of NF␬Bp65. Western blot analysis revealed clear differences in the levels of NF␬Bp65 (Fig. 5A). MSCs from the entire cohort were next assayed for the expression of NF␬Bp65 with the help of computer-based image quantification (Fig. 5B). Separating samples by disease severity revealed a significant difference in nuclear accumulation of NF␬Bp65 among samples of MSCs from preterm infants with mild and severe BPD C low NFκB content 5 high NFκB content #34 (moderate BPD) #20 (severe BPD) #51 #52 proliferation index NFκBp65 Lamin A #20 #54 NFκBp65 Lamin A #34 #32 NFκBp65 4 #51 (moderate BPD) p<0.001 3 #32 (mild BPD) #52 (moderate BPD) 2 Lamin A #54 (mild BPD) 1 0 24 48 72 normalized NFκBp65 expression level A (Fig. 5C). Applying the proportional odds model revealed that high levels of NF␬Bp65 were predictive for the development of severe BPD (Fig. 5D). Biochemical inhibition of the phosphorylation of I␬B␣ confirmed that NF-␬B signaling is important for controlling proliferation in MSCs (Fig. 5E). We next studied additional intracellular markers to detect correlations with the development of severe BPD. We assessed proteins typically expressed in mesenchymal cells including #22 BPD grade severe #24 #29 severe #30 moderate moderate * 2 1.5 1 0.5 96 0 incubation time (hours) B 2.5 #31 #23 #32 severe mild mild internal control mild D 1.0 moderate BPD severe proportional-odds-model; β=2.6 p=0.015 Lamin A NFκBp65 quantification Lamin A quantification relative NFκBp65 95414 72241 45487 61247 31828 18547 39835 69247 254695 237092 249505 269657 185287 177401 248743 218763 0.3746 0.3047 0.1823 0.2271 0.1718 0.1045 0.1601 0.3165 1.1836 0.9627 0.5760 0.7175 0.5428 0.3302 0.5058 1.0000 expression standardized NFκBp65 expression E #62 severe BPD #64 severe BPD 0.8 0.6 severe BPD moderate BPD mild BPD 0.4 0.2 0 0 0.5 1 1.5 2 normalized NFκBp65 expression level -25 * -50 * change in proliferation index (%) 0 estimated probability NFκBp65 -75 #62 Iκκ2 inhibitor IV p-IκBα - #64 + - + IκBα GAPDH AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA ␣-SMA, collagen I␣, myosin heavy chain, and PDGFR-␣. Only the levels of ␣-SMA differed among the three groups (Fig. 6A). MSC samples revealed an inverse cytosolic expression level of ␣-SMA and nuclear NF␬Bp65 (Fig. 6B). As observed for the expression of PI and NF␬Bp65, the expression level of ␣-SMA was distinctly correlated with the degree of BPD severity (Fig. 6C). A high expression level of ␣-SMA was predictive of a good pulmonary outcome (Fig. 6D). The combined analysis of the levels of PI, NF␬Bp65, and ␣-SMA revealed good accuracy of prediction for moderate or severe BPD when logistic regression was used with an area under the curve (AUC) of 0.847 (Fig. 6E). Taken together, the parameters PI, NF␬Bp65 accumulation, and expression of ␣-SMA are useful markers to predict the pulmonary prognosis. Regulation of Proliferative Capacity of MSCs and ␣-SMA Expression by NF␬Bp65 We used RNA interference against NF␬Bp65 to substantiate our findings on the molecular level. The efficient delivery of siRNA against NF␬Bp65 inhibited spontaneous proliferation and led to increased expression of ␣-SMA in MSCs from preterm infants with moderate or severe BPD (Fig. 7). These data suggest that NF␬Bp65 is responsible for the regulation of proliferation and expression of ␣-SMA in MSCs. We next focused on identifying the cause of the accumulation of NF␬Bp65. Alterations in MSCs Characterized by Proinflammatory Cytokines The pulmonary inflammatory response in preterm infants is characterized by an imbalance of proinflammatory cytokines and growth factors. IL-1␤ and TNF-␣ represent important contributors to the inflammatory response in the preterm lung (42, 43). Measurements of IL-1␤ in the supernatant of tracheal aspirates confirmed a positive association between higher levels of IL-1␤ and an increased PI (Fig. 8A). We next mimicked the in vivo environment and stimulated MSCs with recombinant IL-1␤ and TNF-␣. Both cytokines consistently increased the PI in a panel of cultured MSCs (Fig. 8, B–D). Furthermore, both cytokines induced the accumulation of NF␬Bp65 and reduced the expression of ␣-SMA (Fig. 8, B and C). The effect of cytokine stimulation was accompanied by the nuclear translocation of NF␬Bp65 (Fig. 8E). Dose-response analyses revealed gradual transition to an inflammatory phenotype depending on the extent of the proinflammatory stimulus (Fig. 8F). A one-time cytokine stimulation was sufficient to induce L95 persistent alterations in the phenotype of MSCs (Fig. 8G). These data agree with the previous observation indicating that phenotypic alterations in MSCs from preterm infants with severe BPD persisted for several passages under cell culture conditions. Finally, RNA interference, used in the experimental setting shown in Fig. 7, was modified so that the baseline level of nuclear NF␬Bp65 was not affected. Subsequent stimulation with IL-1␤ or TNF-␣ markedly reduced the nuclear accumulation of NF␬Bp65 and the PI after cytokine stimulation (Fig. 9, A–D). DISCUSSION We identified a combination of phenotypic alterations in MSCs isolated from the tracheal aspirates of preterm infants; these MSCs allow for the prediction of better or worse pulmonary prognosis in these children. Surprisingly, we were able to clearly separate children with good and poor pulmonary prognosis in the relatively small patient cohort studied. Molecular studies indicated a link between phosphorylation of I␬B␣, the nuclear accumulation of NF␬Bp65, and the development of severe BPD. The accumulation of NF␬Bp65, induced by IL-1␤ and TNF-␣, was responsible for the increased proliferative capacity of MSCs and was accompanied by the reduced expression of ␣-SMA. Notably, a one-time in vitro stimulation led to persistent alterations in the MSC phenotype lasting for days. These alterations were identical to those observed in MSCs freshly isolated from preterm infants who later developed severe BPD. Taken together, our data clearly indicate that alteration in the MSC phenotype is a critical event in the development of BPD. The data presented here support the dominant role of NF-␬B within the cellular pulmonary inflammatory response; NF-␬B represents a central transcription factor with respect to proliferation and inflammation in many inflammatory diseases (15, 18, 27). High expression levels of NF-␬B within the total cellular fraction of tracheal aspirates are associated with later development of BPD, but the detailed analyses of specific cellular fractions have not yet been conducted. Cellular fractions possess a heterogeneous composition; hence, the predictive value was limited in previous studies (2, 5). Because MSCs represent a very small cellular fraction (data not shown), the determination of the expression level of NF␬Bp65 in MSCs was not achievable in previous studies. Using cell sorting and single cell analyses to optimize the procedure described here will enable early determination of proliferative capacity and expression of NF-␬B and ␣-SMA in the majority of patients as Fig. 6. Protein expression levels in mesenchymal stromal cells (MSCs) correlate with bronchopulmonary dysplasia (BPD) severity and can serve as predictive markers for pulmonary outcome. Reduced expression of ␣-SMA together with an increased PI and augmented nuclear accumulation of NF␬Bp65 can serve to predict severe BPD. The expression level of other proteins showed no differences between MSC cultures. A: using immunofluorescence, MSCs from 2 patients with mild or severe BPD did not differ in the expression levels of PDGF receptor-␣ (PDGFR␣), collagen-I␣, myosin heavy chain, while ␣-SMA expression was reduced in MSCs obtained from the infant with severe BPD. B: in Western blot, ␣-SMA expression was reduced in cytosolic extracts of selected MSCs from preterms with moderate or severe BPD. GAPDH served as loading control. The order of samples in the blot was rearranged without any further manipulation (indicated by separated lines). C: the expression level of ␣-SMA was significantly reduced in preterms with severe BPD in the total cohort when the technique from Fig. 5B was applied to the total cohort. Cytosolic extracts were available from n ⫽ 36 patients. Statistical analysis was performed using one-way ANOVA and post hoc pairwise comparisons by means of t-tests with Bonferroni adjustment. *P ⬍ 0.05 indicates statistical significance. D: predictive accuracy of ␣-SMA was verified using the proportional odds model. P ⫽ 0.018 indicates statistically significant differences. E: the receiver operating characteristic curve (ROC) for combining PI, NF␬Bp65, and ␣-SMA data from Figs. 4A, 5C, and 6C in a logistic model predicted moderate/severe BPD with an area under the curve (AUC) of 0.847. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L96 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA shown before (22, 48). Once this has been achieved, the determination of MSC phenotype may be a promising biomarker for predicting pulmonary outcome and establishing a protocol for early treatment decisions. The general applicabil- A #16 (mild BPD) ity of this biomarker approach requires validation in an independent cohort of patients (13). Inflammation, infection, exposure to mechanical ventilation, and oxygen toxicity are important risk factors in the pathogen- B #22 (severe BPD) BPD grade #13 #12 #23 #26 mild mild mild mild #41 #31 #30 NFκBp65 Lamin A PDGFRα nuclear extracts α-SMA Collagen Iα myosin heavy chain GAPDH Collagen Iα cytosolic extracts normalizedα-SMA expressionlevel C myosin heavy chain α-SMA * 3 2 1 0 mild D moderate BPD severe E 1.0 proportional-odds-model; β=1.07 p=0.018 0.8 0.8 severe BPD moderate BPD mild BPD 0.4 0.2 sensitivity estimated probability 1.0 0.6 0.6 0.4 0.2 AUC: 0,847 0 0 0 0.5 1 1.5 2 2.5 normalizedα-SMA expressionlevel 3 1.0 0.8 0.6 0.4 specifity AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. #29 severe severe moderate moderate 0.2 0 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA 25 mock change proliferation index (%) siNFκBp65 0 -25 -50 -75 P = 0.014 -100 #19 (mild BPD) co mock #22 (severe BPD) siNFκBp65 co mock siNFκBp65 co mock siNFκBp65 NFκBp65 NFκBp65 Lamin A Lamin A nuclear extracts co α-SMA GAPDH mock siNFκBp65 α-SMA GAPDH cytosolic extracts Fig. 7. Using RNA interference to confirm the central role of NF␬Bp65 in alteration of the mesenchymal stromal cell (MSC) phenotype. RNA interference against NF␬Bp65 reduced the proliferation of MSCs from n ⫽ 15 randomly selected preterm infants with moderate or severe bronchopulmonary dysplasia (BPD). Cell growth was assessed by Cellscreen analysis for 72 h starting 24 h after transfection. Western blot analyses for NF␬Bp65 expression were performed 24 h after transfection and for ␣-smooth muscle actin (␣SMA) after 72 h. The calculated relative change in the proliferation index (%) is presented as the mean and 95% confidence interval compared with those of the untreated control group. Statistical significance was tested using a post hoc Bonferroni adjusted pairwise comparison. *P ⫽ 0.014 indicates statistically significant differences. esis of BPD. These factors induce excessive and prolonged secretion of proinflammatory cytokines. Therefore, dysregulation of cytokine and growth factor signaling is attributed to the development of BPD (13, 43). Under physiological conditions, resident pulmonary mesenchymal cells undergo a highly orchestrated process of myofibroblastic differentiation during lung development (24, 28, 40). Previous studies demonstrated substantial alterations in the pathways controlling the differentiation of MSCs into myofibroblasts; these pathways include PDGF receptor-␣, ␤-catenin, and TGF-␤1 signaling in BPD L97 (33, 37, 38, 40). Here we provide molecular evidence that exposure to proinflammatory cytokines leads to a persistent aberrant phenotype, with reduced expression of ␣-SMA, in pulmonary MSCs. This study was not designed to determine the precise origin of these cells from the proximal or distal airways of the immature lung; however, these cells are of pulmonary origin and display a lung-specific phenotype (3). It is possible that the distortion of myofibroblastic differentiation by pulmonary inflammatory response contributes to the distortion of septation and interstitial fibrosis (16, 29, 37, 38, 40). Our results provide a better understanding of how accumulation of NF␬Bp65 misdirects the functions of MSCs. Conversely, NF-␬B signaling is a key pathway and regulator in the regulation of development, growth, and resolution of inflammation (15, 18, 25, 41). Members of the TNF family are an important class of activators of NF-␬B. The downstream effect of TNF family members depends on specific intracellular signal transduction and includes prosurvival functions (1, 10 – 12, 46). In accordance with this, animal studies have clearly demonstrated that a balanced activation status is critical for normal lung development, and that either overstimulation or inhibition of NF-␬B signaling leads to distortion in normal lung development and a BPD-like phenotype (19, 27). A recent study demonstrated a connection between NF-␬B signal transduction and the TGF-␤ pathway, which is another important signaling pathway involved in lung development (11). Not surprisingly, any distortion in the balance of these signaling pathways can lead to augmented lung injury, and can result in increased induction of apoptosis in mesenchymal progenitor cells (11). Therefore, the direct targeting of NF-␬B can further distort lung development (19). However, selective targeting of NF-␬B signaling in MSCs, or identifying decisive downstream signaling pathway(s) that lead to detrimental activity of NF␬B, can yield new therapeutic options. MSCs are readily obtainable from tracheal aspirates of ventilated preterm infants. Studies on these cells can yield further valuable insights into the pathogenesis of BPD (3, 16, 33). Thorough evaluation of the physiological functions of these MSCs and their distortion in the injured lung is prerequisite for developing efficient therapeutic interventions. Our results indicate that distorted proliferation, nuclear accumulation of NF␬Bp65, and reduction in the ␣-SMA content of MSCs are early key events associated with the development of severe BPD. This study shows that future therapeutic approaches, aiming to prevent or reduce the burden of BPD, should include studies on phenotypic alterations in pulmonary MSCs. The following two scenarios should be considered: 1) reversal of the inflammatory MSC phenotype as achieved using Fig. 8. Increase in mesenchymal stromal cell (MSC) proliferation and NF␬Bp65 accumulation mediated by proinflammatory cytokines IL-1␤ and TNF-␣. Proinflammatory cytokines induced the identical changes observed in MSCs from preterm infants with unfavorable pulmonary outcome. A: IL-1␤ (ng/ml) standardized to sIgA (U/ml) determined in tracheal aspirate supernatants correlated to the proliferation index. Supernatants were available from n ⫽ 29 infants. Statistical analysis was performed using linear regression. *P ⬍ 0.05 indicates statistically significant differences. Stimulation of MSCs with IL-1␤ (B, 300 ng/ml) or TNF-␣ (C, 300 ng/ml) increased proliferation and nuclear NF␬Bp65 and reduced the content of cytosolic ␣-smooth muscle actin (␣-SMA). Different sections from the gel (indicated by separated lines) are presented without any further manipulation. Dots indicate the means of at least three different measurements ⫾ SE. D: stimulation of n ⫽ 12 randomly selected cell cultures with IL-1␤ and TNF-␣ increased spontaneous proliferation. Statistical analysis was performed using a linear mixed model. *P ⬍ 0.05 indicates a statistically significant difference vs. control. E: nuclear translocation of NF␬Bp65 was induced after stimulation with IL-1␤ (300 ng/ml) for the time periods indicated. F: increasing the dosage of IL-1␤ from 3 to 300 ng/ml increased cell proliferation in MSCs. G: separation of data from F into 24-h time intervals revealed a persistent increase in proliferation and reduction in ␣-SMA content. Statistical analysis was performed using Student’s t-test. NS, not statistically significant; BPD, bronchopulmonary dysplasia. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L98 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA C A 4.5 #19 (mild BPD) co * TNFα p=0.002 proliferation index 600 IL-1β/sIgA ng/U * 4 400 R2 linear=0.378 200 3.5 * 3 2.5 co TNF-α 2 αSMA GAPDH 1.5 0 0 1 2 1 5 4 3 proliferation index co 0 0,5 24 48 1 72 96 TNF-α 2 6 120 h 12 24 h NFκBp65 Histon H1 B #19 (mild BPD) * 3.5 * 3 2.5 2 co IL-1β αSMA 1.5 GAPDH 1 0 co 24 48 0,5 1 72 96 IL1β 2 6 * D 120 h 12 24 * 3 difference of proliferation index proliferation index * co IL-1β 4 2 1 0 -1 h NFκBp65 -2 Histon H1 IL-1β E #61 (severe BPD) IL-1β co 10min TNFα #62 (severe BPD) 60min co 10min 60min NFκBp65 G Lamin A NFκBp65 GAPDH cytosolic extracts F * 4.5 #19 (mild BPD) proliferation index 4 IL-1β 300ng/ml * 3.5 * 3 * * IL-1β 30ng/ml IL-1β 3ng/ml co * 2.5 % change of proliferation index during time interval nuclear extracts 50 NS #19 (mild BPD) 0 40 NS 48 GAPDH NS 30 NS NS 20 10 0 0-24h 24-48h 48-72h 72-96h 2 1.5 1 24 α-SMA 0 24 48 72 96 120 h AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. 96-120h 72 96 h MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA mock mock+IL-1β siNFκBp65 siNFκBp65+IL-1β 4 * B 3 2 mock mock+TNFα siNFκBp65 siNFκBp65+TNFα 4 proliferation index proliferation index A 24 48 mock + IL-1β NFκBp65 72 96 2 #19 (mild BPD) 0 24 72 96 120 h siNFκBp65 + Histone H1 C D mock mock+IL-1β siNFκBp65 siNFκBp65+IL-1β 4 mock mock+TNFα siNFκBp65 siNFκBp65+TNFα 5 * proliferation index 5 * * 3 * 2 * Fig. 9. Stimulation of mesenchymal stromal cells (MSCs) with pro-proliferative cytokines induces a phenotype that is stable for at least 120 h. Modified RNA interference against NF␬Bp65 before stimulation with IL-1␤ (300 ng/ ml; A and C) or TNF-␣ (300 ng/ml; B and D) reduced the nuclear accumulation of NF␬Bp65 and the PI. The order of Western blot samples was rearranged as indicated by separated lines without any further manipulation. Statistical analysis was performed using ANOVA. *P ⬍ 0.05 indicates statistically significant differences. 4 3 2 #22 (severe BPD) #22 (severe BPD) 1 48 mock + TNF-α NFκBp65 Histone H1 proliferation index 1 120 h siNFκBp65 + * * * 3 #19 (mild BPD) 1 0 L99 0 24 48 72 + siNFκBp65 + mock IL-1β NFκBp65 - Histone H1 96 120 h 1 0 24 48 mock TNF-α NFκBp65 - + 72 96 120 h siNFκBp65 + Histone H1 RNA interference in this study; and 2) prophylactic prevention of phenotypic alterations in MSCs. In addition to the emerging beneficial role of allograft MSCs (35), the crucial role of resident lung MSCs has been discussed with respect to numerous pulmonary disease states of childhood and adolescence (6, 22, 23, 30, 45). Our results encourage future studies to further focus on resident pulmonary MSCs and their role in inflammation and subsequent development of BPD, and to further examine alterations in the MSC phenotype, which account for disease severity. Center Munich Grosshadern and Perinatal Center Giessen. This work is part of the MD theses of T. Reicherzer and S. Häffner. ACKNOWLEDGMENTS AUTHOR CONTRIBUTIONS We thank parents of infants for consent to participate in the study. We gratefully acknowledge the preservation of tracheal aspirates from routine suctioning by the staff of the Neonatal Intensive Care Units at the Perinatal T.R. and H.E. conceived and designed research; T.R., S.H., T.S., J. Gronbach, J.M., and H.E. performed experiments; T.R., S.H., C.H., U.H., J. Gertheiss, A.S., and H.E. analyzed data; T.R., S.H., J.M., J. Gertheiss, and H.E. GRANTS This work was supported by Stiftung Projekt Omnibus, Wilhelm-Vaillant Stiftung (both to H. Ehrhardt), Friedrich-Baur-Stiftung 0030/2008 (to H. Ehrhardt and C. Hübener), and FöFoLe no. 49-2009 (to H. Ehrhardt and A. Schulze). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00505.2017 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung (054.161.069.107) on June 18, 2020. L100 MESENCHYMAL STROMAL CELLS AND BRONCHOPULMONARY DYSPLASIA interpreted results of experiments; T.R., J. Gertheiss, and H.E. prepared figures; T.R. and H.E. drafted manuscript; T.R., J. Gertheiss, S.B., R.E.M., A.H., and H.E. edited and revised manuscript; T.R., S.H., T.S., J. Gronbach, J.M., C.H., U.H., J. Gertheiss, A.S., S.B., R.E.M., A.H., and H.E. approved final version of manuscript. 18. 19. REFERENCES 1. 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