Mesenchymal Stromal Cells Are More Effective Than Their Extracellular Vesicles at Reducing Lung Injury Regardless of Acute Respiratory Distress Syndrome Etiology

Abstract

Although mesenchymal stromal cells (MSCs) have demonstrated beneficial effects on experimental acute respiratory distress syndrome (ARDS), preconditioning may be required to potentiate their therapeutic effects. Additionally, administration of cell-free products, such as extracellular vesicles (EVs) obtained from MSC-conditioned media, might be as effective as MSCs. In this study, we comparatively evaluated the effects of MSCs, preconditioned or not with serum collected from mice with pulmonary or extrapulmonary ARDS (ARDSp and ARDSexp, respectively), and the EVs derived from these cells on lung inflammation and remodeling in ARDSp and ARDSexp mice. Administration of MSCs (preconditioned or not), but not their EVs, reduced static lung elastance, interstitial edema, and collagen fiber content in both ARDSp and ARDSexp. Although MSCs and EVs reduced alveolar collapse and neutrophil cell counts in lung tissue, therapeutic responses were superior in mice receiving MSCs, regardless of preconditioning. Despite higher total cell, macrophage, and neutrophil counts in bronchoalveolar lavage fluid in ARDSp than ARDSexp, MSCs and EVs (preconditioned or not) led to a similar decrease. In ARDSp, both MSCs and EVs, regardless of preconditioning, reduced levels of tumor necrosis factor- (TNF-) α, interleukin-6, keratinocyte chemoattractant (KC), vascular endothelial growth factor (VEGF), and transforming growth factor- (TGF-) β in lung homogenates. In ARDSexp, TNF-α, interleukin-6, and KC levels were reduced by MSCs and EVs, preconditioned or not; only MSCs reduced VEGF levels, while TGF-β levels were similarly increased in ARDSexp treated either with saline, MSCs, or EVs, regardless of preconditioning. In conclusion, MSCs yielded greater overall improvement in ARDS in comparison to EVs derived from the same number of cells and regardless of the preconditioning status. However, the effects of MSCs and EVs differed according to ARDS etiology.

Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this manuscript.

Figures

Figure 1

Schematic flow chart and timeline of study design. ARDS was induced by administration of Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). Control mice (C) received saline solution intratracheally (Cp) or intraperitoneally (Cexp). After 24 h, ARDSp and ARDSexp animals were further randomized to receive saline (50 μL, SAL), bone marrow-derived MSCs (105, 50 μL), or EVs (105, 50 μL), stimulated (MSC serum, EV serum) or not with serum obtained from ARDSp or ARDSexp animals. All data were analyzed on day 2.

Figure 2

Lung histology. Representative photomicrographs of lung parenchyma stained with hematoxylin and eosin from (a) pulmonary ARDS (ARDSp) and (b) extrapulmonary ARDS (ARDSexp) animals. ARDS was induced by administration of Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). Control mice (C) received saline solution intratracheally (Cp) or intraperitoneally (Cexp). After 24 h, ARDSp and ARDSexp animals were further randomized to receive saline (50 μL, SAL), bone marrow-derived MSCs (105, 50 μL), or EVs (105, 50 μL), stimulated (MSC serum, EV serum) or not (MSCs, EVs) with serum obtained from ARDSp or ARDSexp animals.

Figure 3

Total and differential cell counts, as well as protein content, in bronchoalveolar lavage fluid in pulmonary ARDS (ARDSp) (a) and extrapulmonary ARDS (ARDSexp) (b) animals. ARDS was induced by administration of Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). Control mice (C) received saline solution intratracheally (Cp) or intraperitoneally (Cexp). After 24 h, ARDSp and ARDSexp animals were further randomized to receive saline (50 μL, SAL), bone marrow-derived MSCs (105, 50 μL), or EVs (105, 50 μL), stimulated or not with serum (MSCs, EVs, MSC serum, and EV serum) obtained from ARDSp or ARDSexp animals. Values were expressed as mean ± standard deviation of 6 animals per group. ∗Significantly different from the corresponding C group (p < 0.05). ∗∗Significantly different from the corresponding ARDS group (p < 0.05).

Figure 4

Lung mechanics. Static lung elastance (Est, L) and resistive (ΔP1, L) and viscoelastic (ΔP2, L) pressures in animals with experimental pulmonary ARDS (ARDSp) (a) and extrapulmonary ARDS (ARDSexp) (b). ARDS was induced by administration of Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). Control mice (C) received saline solution intratracheally (Cp) or intraperitoneally (Cexp). After 24 h, ARDSp and ARDSexp animals were further randomized to receive saline (50 μL, SAL), bone marrow-derived MSCs (105, 50 μL), or EVs (105, 50 μL), stimulated (MSC serum, EV serum) or not (MSCs, EVs) with serum obtained from ARDSp or ARDSexp animals. Values were expressed as mean + standard deviation of 6 animals per group. ∗Significantly different from the corresponding C group (p < 0.05). ∗∗Significantly different from the corresponding ARDS group (p < 0.05).

Figure 5

Protein levels of mediators in lung tissue. Protein levels of tumor necrosis factor- (TNF-) α, interleukin- (IL-) 6, IL-10, keratinocyte chemoattractant (KC) (a murine IL-8 homolog), vascular endothelial growth factor (VEGF), and transforming growth factor- (TGF-) β in lung tissue homogenate from (a) pulmonary ARDS (ARDSp) and (b) extrapulmonary ARDS (ARDSexp) animals. ARDS was induced by administration of Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). Control mice (C) received saline solution intratracheally (Cp) or intraperitoneally (Cexp). After 24 h, ARDSp and ARDSexp animals were further randomized to receive saline (50 μL, SAL), bone marrow-derived MSCs (105, 50 μL), or EVs (105, 50 μL), stimulated (MSC serum, EV serum) or not with serum (MSCs, EVs) obtained from ARDSp or ARDSexp animals. Boxes show the interquartile (P25-P75) range, whiskers denote the range (minimum-maximum), and the horizontal line represents the median of animals per group. ∗Significantly different from the corresponding C group (p < 0.05). ∗∗Significantly different from the corresponding ARDS group (p < 0.05). #Significantly different from the corresponding MSC group (p < 0.05).

Figure 6

Exposure to conditioned media from MSCs or EVs induces a shift in macrophage polarization in vitro to the M2 rather than the M1 phenotype. Alveolar macrophages (105 cells per well) were collected from (a) Cp and ARDSp or (b) Cexp and ARDSexp mice. Cells were cultured in regular conditions (Cp, Cexp, ARDSp-SAL, and ARDSexp-SAL) or with conditioned media obtained from MSCs (105 cells per well) either unstimulated or stimulated with serum (serum) or extracellular vesicles derived from ARDSp or ARDSexp mice for 24 h. Relative gene expression of iNOS, IL-1β, IL-6, IL-10, arginase, and TGF-β was calculated as a ratio of average gene expression compared to expression of the housekeeping gene 36B4 and presented as fold changes relative to the Cp or Cexp group (alveolar macrophages from Cp or Cexp animals cultured with conditioned media from unstimulated MSCs). Results are presented as means + SD of alveolar macrophages pooled from 3 mice/group. All measurements were performed in triplicate. ∗Significantly different from the corresponding C group (p < 0.05). ∗∗Significantly different from the corresponding ARDS group (p < 0.05). #Significantly different from the corresponding MSC group (p < 0.05).