Multipotent adult progenitor cells prevent functional impairment and improve development in inflammation driven detriment of preterm ovine lungs

Abstract

Background: Perinatal inflammation increases the risk for bronchopulmonary dysplasia in preterm neonates, but the underlying pathophysiological mechanisms remain largely unknown. Given their anti-inflammatory and regenerative capacity, multipotent adult progenitor cells (MAPC) are a promising cell-based therapy to prevent and/or treat the negative pulmonary consequences of perinatal inflammation in the preterm neonate. Therefore, the pathophysiology underlying adverse preterm lung outcomes following perinatal inflammation and pulmonary benefits of MAPC treatment at the interface of prenatal inflammatory and postnatal ventilation exposures were elucidated.

Methods: Instrumented ovine fetuses were exposed to intra-amniotic lipopolysaccharide (LPS 5 mg) at 125 days gestation to induce adverse systemic and peripheral organ outcomes. MAPC (10 × 10⁶ cells) or saline were administered intravenously two days post LPS exposure. Fetuses were delivered preterm five days post MAPC treatment and either killed humanely immediately or mechanically ventilated for 72 h.

Results: Antenatal LPS exposure resulted in inflammation and decreased alveolar maturation in the preterm lung. Additionally, LPS-exposed ventilated lambs showed continued pulmonary inflammation and cell junction loss accompanied by pulmonary edema, ultimately resulting in higher oxygen demand. MAPC therapy modulated lung inflammation, prevented loss of epithelial and endothelial barriers and improved lung maturation in utero. These MAPC-driven improvements remained evident postnatally, and prevented concomitant pulmonary edema and functional loss.

Conclusion: In conclusion, prenatal inflammation sensitizes the underdeveloped preterm lung to subsequent postnatal inflammation, resulting in injury, disturbed development and functional impairment. MAPC therapy partially prevents these changes and is therefore a promising approach for preterm infants to prevent adverse pulmonary outcomes.

Keywords: Bronchopulmonary dysplasia; Postnatal ventilation; Prenatal inflammation; Preterm birth; Stem cell therapy.

Fig. 1

Study design of pre-clinical ovine study for perinatal stress and prenatal MAPC treatment. After the instrumentation at 121 dGA and a recovery period of 4 d, either a bolus of LPS or saline as control (green) was administered i.a. At 127 dGA, the fetus received an i.v. bolus of MAPC (blue) or saline as control. Betamethasone (orange), was administered 24 h before birth and the prenatal cohort received an additional dose 48 h prior to birth (A). At 132 dGA, the premature lamb was delivered via Cesarean section and lambs of the prenatal cohort were humanely killed (A). Lambs of the postnatal cohort were mechanically ventilated after birth (B). After 72 h of MV, the animals of the postnatal cohort were humanely killed (postnatal assessment cohort; B). dGA = days of gestational age, LPS = lipopolysaccharides, MAPC = multipotent adult progenitor cells, MV = mechanical ventilation, i.a. = intra-amniotic, i.v. = intra-venous.

Fig. 2

Perinatal inflammation results in elevated oxygen requirement, which is prevented by MAPC treatment. Prenatal LPS exposure with subsequent postnatal MV led to increased F_IO₂ (A), as well as an increased A-a gradient (B), which was prevented by MAPC treatment (A & B). PIP did not differ between groups (C). Data are presented as mean ± standard deviation. Statistical significance was determined with linear mixed effects models analysis and MV time points were regarded as continuous measurements over 72 h. Significance is indicated by ∗p < 0.05, ∗∗∗p < 0.001. A–a gradient = arterial–alveolar gradient, F_IO₂ = fraction of inspired oxygen, MAPC = multipotent adult progenitor cells, MV = mechanical ventilation, PIP = peak inspiratory pressure.

Fig. 3

Prenatal LPS exposure increases the pulmonary pro-inflammatory immune response at birth and MAPC modulate the immune response to LPS and/or MV in preterm ovine lungs. Prenatal LPS exposure led to increased protein levels of the pro-inflammatory IL-8 in BALf (A). LPS exposure also increased the numbers of CD45⁺ immune cells in the AS of the RLL (B) and of the RUL (C), as well as the IBA-1 immunoreactivity in the AS of the RUL (D). In the AW of the RUL, more IBA-1 immunoreactivity was present in MAPC-treated LPS-exposed animals of the prenatal cohort (E). IL10 mRNA expression levels were unchanged by both LPS exposure and MAPC treatment (F). IL-8 levels did not differ between ventilated groups (G). However, prenatal LPS exposure in combination with MV resulted in higher numbers CD45⁺ immune cells residing in the AS of the RLL (H). MAPC therapy of LPS-exposed animals resulted in a biological relevant increase in the number of CD45⁺ immune cells in the AW following 72 h of MV (I). LPS exposure combined with postnatal MV, resulted in less IBA-1immunoreactivity in both the AS (J) and the AW (K), which was prevented by MAPC treatment. A biological relevant decrease in IL10 mRNA levels was seen in LPS-exposed animals, which was biologically relevant obviated by MAPC therapy (L). Data are presented as median with IQR. IL10 mRNA levels are displayed as relative fold changes over the Sham-Saline group and Sham-Saline-MV group, respectively. Statistical significance was determined with a Kruskal-Wallis test followed by Dunn's multiple comparison and depicted as #p < 0.1, ∗p < 0.05. BALf = bronchioalveolar lavage fluid, AS = alveolar space, AW = alveolar wall, IQR = interquartile range, MAPC = multipotent adult progenitor cells, MV = mechanical ventilation, RLL = right lower lobe, RUL = right upper lobe.

Fig. 4

MAPC modify the redox status in the pulmonary epithelial lining fluid. MAPC therapy following i.a. LPS exposure resulted in a biological relevant increase of the GSH:GSSG ratio compared to untreated animals (A). Also during postnatal assessment, the increase in GSH:GSSG ratio was observed and interestingly, unexposed MAPC-treated animals displayed higher GSH:GSSG levels compared to controls (B). GSH and GSSG concentrations were determined in BALf. Data are presented as median with IQR. Statistical significance was determined with a Kruskal-Wallis test followed by Dunn's multiple comparison and depicted as #p < 0.1, ∗p < 0.05. BALf = bronchioalveolar lavage fluid, i.a. = intra-amniotic, IQR = interquartile range, MAPC = multipotent adult progenitor cells, MV = mechanical ventilation.

Fig. 5

Edema accompanied by cell-junctional loss following perinatal inflammation is counteracted by MAPC treatment and MAPC therapy enhances distal alveolar development. Edema formation was observed in both the RUL (A) and the RLL (B) of preterm ovine lungs, which were exposed to prenatal LPS and postnatal MV. Simultaneously, in MAPC-treated animals, the excess of fluid was reduced (A & B). OCLN expression was disturbed following LPS exposure and normalized after MAPC treatment (C). Similarly, LPS-exposure decreased E-cadherin protein levels normalized for β-actin, which was prevented by MAPC therapy. Cropped protein blots are shown for E-cadherin and β-actin (D). Full-length blots are shown in Supplementary Fig. 2. LPS exposure led to a biological relevant decrease in RAC in the RLL of prenatally assessed animals, which was resolved by MAPC treatment (E). In line, MAPC treatment increased fetal lung TTF-1+ AECs in the presence of prenatal inflammation in the RLL (F). Also in the RUL, MAPC treatment increased fetal lung TTF-1+ AEC2 in the presence of prenatal inflammation compared to controls and untreated animals (G). However, mRNA levels of the distal development driver SOX9 did not differ between groups of the prenatal cohort (H). Postnatally, no changes in RAC (I) and AEC2 (J) were measured. During postnatal assessment, MAPC administration following LPS exposure induced SOX9 mRNA expression in ventilated lambs (K). Data are presented as median with IQR. OCLN and SOX9 mRNA expression and E-cadherin protein levels are displayed as relative fold changes over Sham-Saline-MV. Statistical significance was determined with a Kruskal-Wallis test followed by Dunn's multiple comparison and depicted as #p < 0.1, ∗p < 0.05, ∗∗p < 0.01. IQR = interquartile range, MAPC = multipotent adult progenitor cells, MV = mechanical ventilation, RAC = radial alveolar count, RLL = right lower lobe, RUL = right upper lobe.

Fig. 6

MAPC treatment shifts the immune reaction towards an anti-inflammatory response. Principal component analysis revealed that the second PC separated untreated LPS-exposed lambs from the other groups in the prenatal (A) and postnatal cohort (B). In the prenatal cohort, the LPS-MAPC group clearly clustered along a positive first PC (A), whereas postnatally, this segregation was less prominent (B). Pro-inflammatory vectors were driving the variance between Sham and LPS-exposed groups, which were inversely correlated with developmental, cell junctional and anti-inflammatory vectors contributing to clustering of the LPS-MAPC group in the prenatal study (C) and the postnatal study (D). AS = alveolar space, AW = alveolar wall, MV = mechanical ventilation, PC = principal component, RAC = radial alveolar count, RLL = right lower lobe, RUL = right upper lob.

Supplementary Fig. 1

Edema scoring. Representative histological images are shown for edema score 0 (no edema) (A) and score 3 (alveolar congestion/flooding of alveoli) (B). Image magnification is 200× and for insets 400×. Scale bars: 50 μm; scale bars in insets: 20 μm.

Supplementary Fig. 2

No changes in CD45⁺ immune cells were demonstrated in animals assessed directly at birth for the AW of the RLL (A) and the RUL (B). LPS exposure combined with postnatal MV, did not alter IBA-1immunoreactivity in both the AS (C) and the AW (D) of the RLL. Data are presented as median with IQR. Statistical significance was determined with a Kruskal-Wallis test followed by Dunn's multiple comparison and depicted as #p < 0.1, ∗p < 0.05, ∗∗p < 0.01. AS = alveolar space, AW = alveolar wall, IQR = interquartile range, MAPC = multipotent adult progenitor cells, MV = mechanical ventilation, RLL = right lower lobe, RUL = right upper.

Supplementary Fig. 3

Western blot of E-cadherin and β-actin. Full length Western blot of β-actin at 42kDA (A) and of E-cadherin at 120kDA (B). Crop-positions are indicated within full-length blots with rectangles.

Supplementary Fig. 4

MAPC therapy following antenatal LPS exposure increases the number of AEC2. RAC of the RUL were not different between groups neither for the prenatal cohort (A) nor for the postnatal cohort (B). However, MAPC treatment increased fetal lung TTF-1+ AEC2 in the presence of prenatal inflammation in the RUL compared to controls and untreated animals (C). In MAPC-treated ventilated animals, the number of TTF-1+ AEC2 cells was biologically relevant decreased following intrauterine LPS exposure (D). Data are presented as median with IQR. Statistical significance was determined with an Kruskal-Wallis test followed by Dunn's multiple comparison and depicted as #p < 0.1, ∗p < 0.05, ∗∗∗p < 0.001. IQR = interquartile range, MAPC = multipotent adult progenitor cells, MV = mechanical ventilation, RAC = radial alveolar count, RUL = right upper lobe.