Therapeutic Effects of Human Mesenchymal Stem Cell-derived Microvesicles in Severe Pneumonia in Mice

Abstract

Rationale: Microvesicles (MVs) are anuclear fragments of cells released from the endosomal compartment or shed from surface membranes. We and other investigators demonstrated that MVs released by mesenchymal stem cells (MSCs) were as effective as the cells themselves in inflammatory injuries, such as after endotoxin-induced acute lung injury. However, the therapeutic effects of MVs in an infectious model of acute lung injury remain unknown.

Objectives: We investigated the effects of human MSC MVs on lung inflammation, protein permeability, bacterial clearance, and survival after severe bacterial pneumonia.

Methods: We tested the effects of MVs derived from human MSCs on Escherichia coli pneumonia in mice. We also studied the interactions between MVs and human monocytes and human alveolar epithelial type 2 cells.

Measurements and main results: Administration of MVs derived from human MSCs improved survival in part through keratinocyte growth factor secretion and decreased the influx of inflammatory cells, cytokines, protein, and bacteria in mice injured with bacterial pneumonia. In primary cultures of human monocytes or alveolar type 2 cells, the uptake of MVs was mediated by CD44 receptors, which were essential for the therapeutic effects. MVs enhanced monocyte phagocytosis of bacteria while decreasing inflammatory cytokine secretion and increased intracellular ATP levels in injured alveolar epithelial type 2 cells. Prestimulation of MSCs with a toll-like receptor 3 agonist further enhanced the therapeutic effects of the released MVs.

Conclusions: MVs derived from human MSCs were as effective as the parent stem cells in severe bacterial pneumonia.

Keywords: acute respiratory distress syndrome; bacterial pneumonia; mesenchymal stem cells; microvesicles.

Figure 1.

Characterization of microvesicles (MVs) released from human mesenchymal stem cells (MSCs) (MSC MVs). (A) After serum starvation, human MSCs constitutively release MVs (arrows), small-membrane enclosed bodies from the plasma membrane (scale bar = 1 μm). Insert shows purified MSC MVs as a homogeneous population of spheroid particles (scale bar = 2 μm). (B) The protein concentration of the therapeutic dose, 90 μl of MSC MVs, used for the small animal studies was similar to the dose of MSC MVs previously used in the literature. MSC MVs contained a significant amount of total RNA. MSC MV protein and RNA contents are expressed as mean ± SD total protein (μg/90 μl) and total RNA (ng/90 μl), respectively (n = 24 for protein; n = 7 for RNA content). (C) Western blot analyses of MSC MVs demonstrated significant levels of CD44, a receptor previously involved in MSC trafficking to inflammatory sites. Similar to MSCs, we hypothesized that MSC MVs homed to injured tissues using the CD44 receptor. MW = molecular weight.

Figure 2.

Keratinocyte growth factor (KGF)-mediated effect of mesenchymal stem cell (MSC) microvesicles (MVs) on survival after severe Escherichia coli pneumonia in mice. Intravenous administration of MVs released from MSCs (MSC MVs) improved survival in mice with severe E. coli pneumonia. Administration of a neutralizing KGF antibody with MSC MVs abrogated this therapeutic effect. (A) Administration of MSCs or MSC MVs significantly increased survival over 72 hours. *P < 0.01 versus phosphate-buffered saline (PBS), ^#P < 0.05 versus normal human lung fibroblast (NHLF) MV-treated group, and ^†P < 0.05 versus PBS by log-rank test. (B) Reverse transcriptase polymerase chain reaction demonstrated that MSC MVs expressed KGF messenger RNA. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control to normalize loading of the RNA samples. The PCR products were 210 bp in size for KGF. (C) Intravenous administration of MSC MVs increased human KGF protein levels in the bronchoalveolar lavage fluid (BAL) of E. coli–injured mice at 18 hours, which was not accounted for by MV intracellular KGF protein levels; lysates of 90 μl of MVs yielded only 16.5 ± 5.4 pg total of KGF protein. Data are shown as mean ± SD for each condition (n = 29 for PBS, n = 12 for MSCs, n = 22 for MVs released from standard mesenchymal stem cells, and n = 10 for polyinosine-polycytidylic acid [Poly (I:C)] MVs). ***P < 0.01 versus the Poly (I:C) MV group by ANOVA (Bonferroni). (D) Administration of an anti-KGF neutralizing antibody with the MSC MVs significantly decreased survival over 72 hours compared with the MSC MV + control IgG–treated group. *P < 0.01 versus PBS; ^✔P < 0.01 versus MSC MV + anti-KGF antibody–treated group by log-rank test. MV + IgG = MVs released from MSCs combined with control IgG antibody; MV + anti-KGF Ab = MVs released from MSCs combined with an anti-KGF neutralizing antibody; NHLF MV = MVs released from normal human lung fibroblasts.

Figure 3.

Effect of microvesicles (MVs) released from mesenchymal stem cells (MSCs) (MSC MVs) on the influx of inflammatory cells and on inflammation after Escherichia coli–induced acute lung injury in mice. Intravenous and intratracheal administration of human MSC MVs reduced the influx of inflammatory cells and lung protein permeability in the injured alveoli after E. coli pneumonia. (A) Intravenous administration of MSC MVs decreased total white blood cell (WBC) and neutrophil counts in the bronchoalveolar lavage (BAL) fluid of E. coli–injured mice at 24 hours. Data are shown as mean ± SD for each condition (n = 21 for sham, n = 20 for phosphate-buffered saline (PBS), n = 6 for MSCs, n = 22 for MSC MVs, and n = 5 for normal human lung fibroblast [NHLF] MVs). *P < 0.05 versus PBS by ANOVA (Bonferroni) for neutrophil count, **P < 0.01 versus PBS; ^✔P < 0.05 versus NHLF MV group by ANOVA (Bonferroni) for total WBCs. (B) Intravenous MSC MVs reduced lung protein permeability and MIP-2 levels in BAL fluid of mice injured with E. coli pneumonia at 24 and 18 hours, respectively (n = 17–21 for sham, n = 19–22 for PBS, n = 6–9 for MSCs, n = 14–20 for MSC MVs, and n = 5–14 for NHLF MVs). *P < 0.05 versus MSCs for protein concentration by ANOVA (Bonferroni), ***P < 0.01 versus PBS, and ^✔P < 0.05 versus NHLF MVs. (C) Intratracheal MSC MV administration decreased total WBCs, neutrophil counts, and the protein concentration in the BAL fluid of E. coli–injured mice at 24 hours. Data are shown as mean ± SD for each condition (n = 2–3 for sham, n = 5–20 for PBS, n = 9–14 for MSC MVs, and n = 5 for NHLF MVs). MIP-2 = macrophage inflammatory protein-2.

Figure 4.

Effect of the administration of microvesicles (MVs) released from mesenchymal stem cells (MSCs) (MSC MVs) on lung injury after severe Escherichia coli pneumonia in mice. (A) Intravenous MSC MVs or MSCs significantly improved lung injury as assessed by histology. Hematoxylin and eosin–stained lung sections at 18 hours exhibited a reduction in neutrophil influx, edema, wall thickening, and airspace congestion. (B) Lung injury as assessed by semiquantitative scoring was reduced by MSC or MSC MV treatment. Data are shown as mean ± SD for each condition (n = 4–5). *P < 0.01 versus phosphate-buffered saline (PBS), ^✔P < 0.01 versus the normal human lung fibroblast (NHLF)-treated group, and ^†P < 0.01 versus MSCs by Kruskal-Wallis test (Dunn).

Figure 5.

Effect of microvesicles (MVs) released from mesenchymal stem cells (MSCs) (MSC MVs) on total bacterial load. Intravenous administration of human MSC MVs reduced the total bacterial load in the alveolar space (A), lung tissue (B), and bloodstream (C) after Escherichia coli pneumonia. Intratracheal administration of human MSC MVs numerically reduced the total alveolar bacterial load (D) and significantly reduced the incidence of bacteremia (E). (A) Intravenous MSC MVs decreased the total alveolar bacterial load in mice injured by E. coli pneumonia at 18 hours. Total bacterial counts were expressed as mean (colony-forming unit [cfu] counts/ml) ± SD for each condition (n = 15 for phosphate-buffered saline [PBS], n = 9 for MSCs, n = 11 for MSC MVs, and n = 7 for normal human lung fibroblast [NHLF] MVs). *P < 0.05 versus PBS; ^✔P < 0.05 versus NHLF MV group by Kruskal–Wallis test (Dunn). (B) Intravenous MSC MVs decreased the total bacterial load in the lung homogenate of mice injured by E. coli pneumonia at 18 hours. Total bacterial counts were expressed as mean (cfu counts/ml) ± SD for each condition (n = 9 for PBS; n = 8 for MSC MVs). *P < 0.05 versus PBS group by Mann–Whitney test. (C) Compared with PBS, the MSC MV–treated group was without bacteremia at 18 hours. Total bacterial counts were expressed as individual plotted value (cfu counts/ml) (the bar represents the median for each condition; n = 10 for both groups). (D) Intratracheal MSC MVs numerically decreased total bacterial load in the injured alveolus and decreased the bacteria load in the blood of mice injured by E. coli pneumonia at 24 hours. (E) Total bacterial counts were expressed as individual plotted value (cfu counts/ml) (the bar represents the median for each condition; n = 5 for PBS, n = 9 for MSC MVs, and n = 5 for MVs released from NHLFs [NHLF MVs]). BAL = bronchoalveolar lavage.

Figure 6.

Role of CD44 in the uptake of microvesicles (MVs) released from mesenchymal stem cells (MSCs) (MSC MVs) into primary cultures of human monocytes and human alveolar epithelial type 2 cells. In both human monocytes and human alveolar epithelial type 2 cells, MSC MV uptake was dependent on CD44, the cell surface receptor for hyaluronic acid, after injury. (A) LPS stimulation increased the uptake of fluorescent-labeled MSC MVs into monocytes, which was dependent on the CD44 receptor on the MVs. Fluorescence intensity was expressed as mean (arbitrary units) ± SD for each condition (n = 352–477 cells for all groups). *P < 0.01 versus LPS−, ^†P < 0.01 versus CD44-preincubated MSC MVs, and ^§P < 0.01 versus IgG preincubated MSC MVs by ANOVA (Bonferroni). Photomicrographs display the pattern of fluorescence levels observed in each experimental condition. Scale bar = 20 μm. (B) Using MVs released by green fluorescent protein–transfected MSCs, we confirmed that LPS stimulated the uptake of MSC MVs (n = 269–343). *P < 0.01 versus LPS− by Student’s t test. Photomicrographs display the pattern of fluorescence levels (green fluorescent protein) observed in both experimental conditions. Scale bar = 20 μm. (C) Stimulation by LPS with an inflammatory injury (cytomix + LPS) increased the uptake of fluorescent-labeled MSC MVs into alveolar epithelial type 2 cells, which was dependent on the CD44 receptor on the MVs. Fluorescence intensity was expressed as mean (arbitrary units [A.U.]) ± SD for each condition (n = 179–239 cells for all groups). *P < 0.01 versus cytomix− LPS−, ^†P < 0.01 versus CD44-preincubated MSC MVs, and ^§P < 0.01 versus IgG preincubated MSC MVs by ANOVA (Bonferroni). Photomicrographs display the pattern of fluorescence levels observed in each experimental condition. Scale bar = 20 μm. (D) Blocking the CD44 receptor on MSC MVs decreased survival in mice injured with Escherichia coli pneumonia treated with the MVs as compared with blocking with an IgG control antibody (n = 21 for MV + IgG, n = 33 for MV + CD44ab). *P < 0.01 versus MV + IgG by log-rank test. Ab = antibody.

Figure 7.

Functional effects of microvesicles (MVs) released by mesenchymal stem cells (MSCs) (MSC MVs) on primary cultures of human monocytes and human alveolar epithelial type 2 (ATII) cells. (A) Treatment of human blood monocytes with MSC MVs for 24 hours increased Escherichia coli bacterial clearance by 24%. Total bacterial counts are expressed as mean (% of phosphate-buffered saline [PBS]) ± SD for each condition (n = 46 for PBS, n = 12–13 for MVs released by normal human lung fibroblasts [NHLFs] [NHLF MVs] or MSCs, and n = 26 for MSC MVs). *P < 0.05 versus PBS, **P < 0.01 versus PBS, and ^✔P < 0.01 versus NHLF MVs by ANOVA (Bonferroni). (B) MSC MVs decreased monocyte tumor necrosis factor α (TNF-α) secretion by 30%. TNF-α secretion levels are expressed as mean (% of PBS) ± SD for each condition (n = 21 for PBS, n = 10 for NHLF MVs or MSCs, and n = 15 for MSC MVs). ***P < 0.01 versus PBS, ****P < 0.01 versus PBS, and ^✔P < 0.01 versus MSCs by ANOVA (Bonferroni). (C) MSC MVs restored intracellular ATP levels in human ATII cells injured with an inflammatory insult (cytomix) at 48 hours (n = 11–14 per condition).

Figure 8.

Effect of mesenchymal stem cell (MSC) pretreatment with polyinosine-polycytidylic acid [Poly (I:C)], a toll-like receptor 3 (TLR3) agonist, on messenger RNA (mRNA) expression for cyclooxygenase 2 (COX2) and IL-10 in MSCs and its released microvesicles (MVs) and in monocytes exposed to MVs released by Poly (I:C)–pretreated MSCs (MSC MVs). In these experiments, total RNA was extracted from human MSCs (B, C), MSC MVs (D), or human monocytes (E, F), and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed. Top: Representative agarose gels of semiquantitative RT-PCR products from COX2 (B, D, E) and IL-10 (C, F) mRNA amplification. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control to normalize loading of the RNA samples. The PCR products were 295 bp in size for COX2 and 500 bp for IL-10. Bottom: Densitometry readings derived from the PCR gels. The band density relative to that of the GAPDH is expressed as mean ± SD. (A) Human MSCs expressed TLR3. (B, C) Human MSCs were cultured with or without Poly (I:C) for 1 hour and serum starved for 48 hours. TLR3 stimulation further increased the expression of COX2 and IL-10 mRNA in MSCs [n = 8–22 for standard control (STD MSCs) and Poly (I:C)–stimulated MSCs]. *P < 0.01 by Student’s t test. (D) TLR3 stimulation also further increased the mRNA expression for COX2 in the released MVs, Poly (I:C) MVs (n = 10 for MVs released from standard MSCs [STD MVs] and MVs released from MSCs prestimulated with Poly (I:C) MVs [Poly (I:C) MVs]). *P < 0.01 by Mann–Whitney test. (E, F) mRNA expression for COX2 and IL-10 was increased in human monocytes exposed to Poly (I:C) MVs (n = 5–8). *P < 0.01 by Student’s t test.

Figure 9.

Monocytes exposed to microvesicles (MVs) derived from polyinosine-polycytidylic acid [Poly (I:C)]–stimulated mesenchymal stem cells (MSCs) had increased bacterial phagocytosis index and decreased inflammatory cytokine secretion. (A–C) Representative phagocytosis of Escherichia coli bacteria by human monocytes exposed to phosphate-buffered saline (PBS) (A, control), MVs released from standard MSCs (STD MVs) (B), or MVs released from MSCs prestimulated with Poly (I:C) [Poly (I:C) MVs] (C). Scale bar = 5 μm. (D–F) Human monocytes exposed to Poly (I:C) MVs exhibited a higher E. coli bacterial clearance capacity compared with STD MV treatment (D). Although the phagocytosis rate was similar between groups (E), treatment with Poly (I:C) MVs further increased the phagocytosis index for bacteria compared with treatment with STD MVs (F). Data are expressed as mean ± SD [n = 5–13 for PBS, n = 6–22 for STD MVs, and n = 6–19 for Poly (I:C) MVs]. ***P < 0.01 versus PBS; ^✔P < 0.01 versus STD MVs by ANOVA (Bonferroni). (G, H) Poly (I:C) MVs further decreased tumor necrosis factor α (TNF-α) and increased IL-10 levels secretion by monocytes [n = 11–13 for PBS, n = 11–12 for STD MVs, and n = 11–20 for Poly (I:C) MV]. **P < 0.01 versus PBS, ***P < 0.01 versus PBS, and ^✔P < 0.01 versus STD MVs by ANOVA (Bonferroni). (I) In mice injured with E. coli pneumonia, intravenous administration of Poly (I:C) MVs further decreased the alveolar bacterial load as compared to STD MSC MVs. Total bacterial counts are expressed as mean (% of STD MSC MVs) ± SD for each condition [n = 14 for STD MSC MVs; n = 8 for Poly (I:C) MVs]. BAL = bronchoalveolar lavage.