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Mesenchymal Stem Cell–Derived Extracellular Vesicles Alleviate Acute Lung Injury Via Transfer of miR-27a-3p*

Wang, Jiangmei MD1; Huang, Ruoqiong MD1; Xu, Qi MD1; Zheng, Guoping MD2; Qiu, Guanguan MS2; Ge, Menghua MD2; Shu, Qiang MD1; Xu, Jianguo PhD1,2

Author Information
doi: 10.1097/CCM.0000000000004315

Abstract

Despite improvements in lung-protective ventilation and fluid management, the mortality of acute respiratory distress syndrome (ARDS, acute lung injury) still remains as high as 30–40% (1). Alternative therapeutic methods are urgently needed because none of the current pharmacologic interventions is effective for ARDS. Many studies, including observations from our group (2), have documented that mesenchymal stem cells (MSCs) are capable of alleviating acute lung injury induced by lipopolysaccharide or live bacteria in mice (3–5). In addition, clinical trial results from our group (6) and the others (7) showed that allogenic MSCs are safe for short term and well tolerated by ARDS patients. The therapeutic benefits of MSCs in animals are proposed to be mediated by extracellular vesicles (EVs) (8) and soluble paracrine factors such as keratinocyte growth factor (KGF) (9), antimicrobial peptides (10), and angiopoietin-1 (4).

EVs are a heterogeneous population of spherical membrane structures released by almost all types of cells. These vesicles consist of the lipid bilayer with transmembrane proteins and contain cellular components such as lipids, cytosolic proteins, DNA, RNA, and microRNAs (miRNAs). EVs serve as a novel mechanism of intercellular communication by transferring cellular components between cells (11). According to the most recent statement from the International Society for Extracellular Vesicles (ISEV), both EV and non-EV markers should be examined to determine the presence of EVs (12). EV markers include transmembrane proteins and cytosolic proteins, which are incorporated into EVs via membrane binding. EVs are traditionally classified into three subtypes according to sizes and biogenesis mechanisms: exosomes (50–150 nm), microvesicles (100–1,000 nm), and apoptotic bodies (500–5,000 nm). ISEV has now categorized EVs as small EVs (< 100 or < 200 nm) and medium/large EVs (> 200 nm) based on physical characteristics (12). MSC-EVs have similar therapeutic effects as MSCs and offer several advantages, including low immunogenicity and simple storage (13). Therefore, MSC-EVs may serve as an alternative to whole-cell therapy for the treatment of a plethora of diseases.

miRNAs are small noncoding 18- to 25-nucleotide RNAs that interfere with protein translation by binding to complementary nucleotide of target messenger RNA (mRNA), typically in the 3′ untranslated region (UTR) (14). Transfer of miR-125a from MSC-EVs to endothelial cells mediated cell-to-cell communication and promoted angiogenesis (15). EVs from glioma-associated MSCs enhanced the proliferation of stem-like cells in glioma via transfer of miR-1587 (16). Furthermore, MSC-EVs facilitated the miR-133b transfer to neurons and enhanced functional recovery after stroke in rats (17). Therefore, it is possible that miRNA transfer from MSC-EVs might participate in alleviating acute lung injury.

Zhu et al (18) reported that MSC-EVs ameliorated lipopolysaccharide-induced acute lung injury via KGF. The same group also discovered that MSC-EVs alleviated lung injury in an ex vivo–perfused human model of bacterial pneumonia (19). Tang et al (20) documented that MSC-EVs attenuated lipopolysaccharide-induced lung injury via angiopoietin-1 mRNA. Our group recently found that young and aging MSC-EVs have differential effects in lipopolysaccharide-induced lung injury. In the present study, we aimed to determine whether transfer of miRNA from MSC-EVs to alveolar macrophages played a role in acute lung injury. Our results revealed that miR-27a-3p was transferred from MSC-EVs to alveolar macrophages, modulated macrophage polarization, and alleviated acute lung injury.

MATERIALS AND METHODS

Full details are available in the supplemental material (Supplemental Digital Content 1, http://links.lww.com/CCM/F377).

Isolation of MSC-EVs

MSCs were cultured in Dulbecco's Modified Eagle Medium low glucose supplemented with EV-depleted fetal bovine serum obtained from ultracentrifugation at 118,000g for 16 hours at 4°C (approximately 80% of serum EVs was depleted after ultracentrifugation as assayed via nanoparticle tracking analysis). The culture medium was collected and centrifuged sequentially for 15 minutes at 1,500g and 30 minutes at 16,500g, followed by centrifugation at 118,000g for 2 hours at 4°C. Protein concentration of EVs was determined by a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA).

Treatment of Mouse Bone Marrow–Derived Macrophages

Bone marrow–derived macrophages (BMDMs) were cultured alone or cultured with MSC-EVs (100 µg/mL) and sim ultaneously stimulated with or without lipopolysaccharide (100 ng/mL) for 24 hours. Then, BMDMs were harvested for quantitative real-time polymerase chain reaction (qRT-PCR) analysis.

Transducing Anti-miR-27a-3p Into MSCs and BMDMs

Anti-mouse-miR-27a-3p and control anti-miR Lentivirus were purchased from GeneChem (Shanghai, China). Primary BMDMs and human MSCs were infected with Lentivirus (multiplicity of infection of 60). BMDMs were then cultured with MSC-EVs (100 µg/mL) and simultaneously stimulated with lipopolysaccharide (100 ng/mL) for 24 hours. After that, BMDMs were harvested for qRT-PCR analysis. MSC-EVs from transduced MSCs were prepared as described earlier and used for in vivo experiments.

Microarray Analysis

THP-1 cells were cultured alone or cocultured with MSCs via Transwell (Corning, New York, NY) and treated with lipopolysaccharide (100 ng/mL) for 24 hours. Total RNA was then extracted from THP-1 cells. Microarray experiments were assayed using the miRCURY LNA Array platform (Exiqon, Vedbaek, Denmark). Data were analyzed via GenePix Pro 6.0 software (Molecular Devices, San Jose, CA). The threshold value to define an up-regulation of miRNAs was a fold change of 1.5 with a p value of less than 0.1. Finally, heat map was performed to show distinguishable miRNA expression profiling among samples.

Model of Lipopolysaccharide-Induced Lung Injury

C57BL/6 mice (6–8 wk old) were used in all experiments. Protocols for animal research were preapproved by the Institutional Animal Care and Use Committee of Zhejiang University School of Medicine. Mice were randomly divided into five groups: control, lipopolysaccharide, lipopolysaccharide + MSCs IV, lipopolysaccharide + EVs IV, and lipopolysaccharide + EVs intratracheally. Thirty minutes after lipopolysaccharide treatment (5 mg/kg of lipopolysaccharide intratracheally), 1 × 106 cells, 50 µg of EVs, or phosphate-buffered saline were administered via the tail veins of the mice. In another group, 50 μg of EVs was administered via intratracheally. Lungs and bronchoalveolar lavage (BAL) samples were collected for analysis after 48 hours.

Dual Luciferase Assay

For dual luciferase assay, a 206 base pair 3′UTR segment and a mutant segment of nuclear factor kappa B subunit 1 (NFKB1) corresponding to the putative binding site of miR-27a-3p were synthesized and inserted into the pGL3 reporter vector. pGL3-NFKB1 3′UTR or mutant pGL3-NFKB1 3′UTR was cotransfected with pcDNA-miR-27a-3p or empty pcDNA vector into human embryonic kidney 293 (HEK293) cells. Luciferase activities were measured 48 hours later.

Statistical Methods

Data are presented as mean ± sd. Student t test or one-way analysis of variance with Bonferroni post hoc analysis was performed for parametric data with an n value of greater than or equal to 8. The Mann-Whitney test or Kruskal-Wallis test with Dunn post hoc analysis was performed for small sample size (n < 8) and nonparametric data with an n value of greater than or equal to 8. Data were tested for normality by using the D′Agostino-Pearson Omnibus normality test, the Shapiro-Wilk normality test, and Kolmogorov-Smirnov test with Dallal-Wilkinson-Lilliefor corrected p value. Statistical analysis was carried out using the GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA). Results were considered significant if p value was less than 0.05.

RESULTS

Uptake of MSC-EVs by Monocytes/Macrophages In Vitro and In Vivo

Transmission electron microscopy (TEM) analysis revealed that MSC-EVs had a cup-shaped morphology with a diameter of 50–150 nm in size (Supplemental Fig. 1A, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). Their size distribution as determined via dynamic light scattering analysis showed a peak at approximately 106 nm in diameter (Supplemental Fig. 1B, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379), indicating that MSC-EVs were composed by both microvesicles and exosomes. The ratio of the number of EV particles (determined via nanoparticle tracking analysis) to protein content was 2.36 ± 0.63 × 108 particles/µg, which has been suggested as an indicator of EV purity (21). In medium supplemented with EV-depleted serum, the concentration of MSC-EVs reached 417 ± 156 particles per cell at 24-hour post subculture. MSC-EVs expressed markers for exosomes (CD63 and CD81), MSCs (CD105 and CD44), and microvesicles (CD40) in Western blots. Furthermore, MSC-EVs were negative for GM130 (Golgi marker) and calnexin (endoplasmic reticulum marker) (Supplemental Fig. 1C, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379).

To study the uptake of MSC-EVs by monocytes/macrophages in vitro, THP-1 monocytes were cultured with or without CD63-labeled MSC-EVs. Confocal microscopy results showed that THP-1 cells procured labeled MSC-EVs (Fig. 1A). Similar experiments were also performed by culturing CD63-labeled MSC-EVs with BMDMs and examined via flow cytometry assay. More than 40% of BMDMs were positive for CD63 (F4/80+CD63+) after 6 hours (Fig. 1B). To determine EV uptake by macrophages in vivo, MSC-EVs were labeled with the fluorescent dye DID and administered intratracheally to mice with lipopolysaccharide-induced lung injury. Flow cytometry results showed that approximately 70% of BAL macrophages were F4/80+DID+ after 24 hours (Fig. 1C).

Figure 1.
Figure 1.:
Mesenchymal stem cell–derived extracellular vesicles (MSC-EVs) were efficiently taken up by monocytes/macrophages. A, MSC-EVs were labeled with or without anti-CD63 antibody for overnight and cultured with THP-1 cells for 24 hr. The cell nucleuses were stained with Hoechst (ImmunoChemistry Technologies, Bloomington, MN). After washing, the samples were subjected to confocal microscopy analysis. B, Bone marrow–derived macrophages were incubated with CD63-labeled extracellular vesicles (EVs) for 6 hr and stained with PE-Cy7-conjugated anti-mouse F4/80. F4/80+CD63+ macrophages were assayed via flow cytometry. C, Mice were treated with lipopolysaccharide via intratracheally and administered with DID-labeled EVs via intratracheally 30 min later. Total cells from bronchoalveolar lavage (BAL) were harvested after 24 hr and assayed for F4/80+DID+ macrophages via flow cytometry. Mann-Whitney U test was used for analysis. Data are presented as mean ± sd; n = 4. *p < 0.05. SSC = side scatter.

Transfer of miR-27a-3p From MSCs and MSC-EVs to Monocytes/Macrophages In Vitro

miRNAs are transferred between cells as a way of intercellular communication. To explore the transfer of miRNAs between MSCs and monocytes/macrophages in vitro, human THP-1 monocytes were cultured alone (lipopolysaccharide) or cocultured with human MSCs (lipopolysaccharide + MSCs) via Transwell in the presence of lipopolysaccharide. miRNA expression in THP-1 cells was compared between the two groups after microarray analysis. Figure 2A showed 10 of the most highly expressed miRNAs with greater than 1.5-fold increase after coculture with MSCs. Although miR-1260b was on the top of the list, it was documented as a cancer-related miRNA (22). miR-27a-3p was the second most highly expressed miRNA in the list and has been reported to participate in M2 macrophage polarization (23,24). In addition, miR-27a-3p has been shown as one of the major miRNAs in MSC-EVs (25). Therefore, we focused our study on miR-27a-3p. Elevated levels of miR-27a-3p in THP-1 cells with coculture of MSCs were validated by qRT-PCR. Our results showed that lipopolysaccharide treatment did not alter the expression of miR-27a-3p in THP-1 cells (Fig. 2B). miR-27a-3p was detected in MSCs, and the levels were significantly higher than those of THP-1 cells, suggesting a transfer of miR-27a-3p from MSCs to THP-1 monocytes (Fig. 2C). To determine whether MSC-EVs mediate the transfer of miR-27a-3p, THP-1 cells were cultured in the presence or absence of MSC-EVs. MSC-EVs mimicked the effect of MSCs in up-regulation of miR-27a-3p levels (Fig. 2D). Human and murine miR-27a-3p sequences share 100% homology. EV transfer of miR-27a-3p was then performed in mouse BMDMs. In contrast to THP-1 cells, lipopolysaccharide down-regulated the expression of miR-27a-3p in BMDMs (Fig. 2E). This result is consistent with previous reports that differentiated and undifferentiated THP-1 cells respond differently to lipopolysaccharide (26). miR-27a-3p levels in BMDMs were increased when cells were treated with MSC-EVs but not EVs from unconditioned medium with or without lipopolysaccharide treatment (Fig. 2E). In addition, pri-miR-27a levels in BMDMs as determined via qRT-PCR analysis were unchanged when incubated with MSC-EVs, indicating a direct transfer of miR-27a-3p rather than a transcriptional up-regulation. Contrarily, lipopolysaccharide decreased miR-27a-3p expression in BMDMs via transcriptional mechanism as reflected by reduced pri-miR-27a levels (Fig. 2F).

Figure 2.
Figure 2.:
miR-27a-3p is transferred from mesenchymal stem cells (MSCs) and MSCs–derived extracellular vesicles (MSC-EVs) to monocytes/macrophages in vitro. A, THP-1 cells were treated with lipopolysaccharide (LPS) (100 ng/mL) or cocultured with MSCs in the presence of LPS (LPS + MSCs) for 24 hr via Transwell. Then, microRNA (miRNA) profile in THP-1 cells was determined via microRNA array and compared between the two groups (n = 3). Ten of the most highly expressed miRNAs demonstrated greater than 1.5-fold increase with a p value of less than 0.1 after MSC coculture were selected for hierarchical cluster analysis to generate the heat map. Green and red on the heat map represent a decrease and increase of miRNA expression, respectively, with color intensities corresponding to relative expression levels. B, THP-1 cells were cultured alone or with MSCs in the presence or the absence of LPS (100 ng/mL) for 24 hr. miR-27a-3p expression in THP-1 cells was determined via quantitative real-time polymerase chain reaction (qRT-PCR). C, miR-27a-3p levels among THP-1 cells, bone marrow–derived macrophages (BMDMs), and MSCs were assayed via qRT-PCR. D, THP-1 cells were cultured alone or with MSC-EVs (100 µg/mL) in the presence or the absence of LPS (100 ng/mL) for 24 hr. miR-27a-3p expression in THP-1 cells was determined via qRT-PCR. E, BMDMs were incubated alone, with MSC-EVs (100 µg/mL), or extracellular vesicles (EVs) from unconditioned medium (UcM; equal volume of medium) in the presence or the absence of LPS (100 ng/mL) for 24 hr and assayed for miR-27a-3p via qRT-PCR. F, BMDMs were incubated alone, with MSC-EVs (100 µg/mL), EVs from UcM (equal volume of medium), or LPS (100 ng/mL) for 24 hr and assayed for pri-miR-27a via qRT-PCR. One-way analysis of variance with Bonferroni post hoc test (D and E) or Kruskal-Wallis test with Dunn post hoc test (B, C, and F) was used for the analysis. Data are presented as mean ± sd; n = 6–8. *p < 0.05, **p < 0.01, and ***p < 0.001.

Role of miR-27a-3p in Mediating the Effect of MSC-EVs on Macrophage Polarization In Vitro

To determine the effect of MSC-EVs on macrophage polarization, BMDMs were cultured alone, with lipopolysaccharide, or with MSC-EVs plus lipopolysaccharide for 24 hours. Then, total RNA was extracted from BMDMs and subjected to qRT-PCR analysis. The increase in mRNA levels of proinflammatory interleukin-1β and tumor necrosis factor (TNF)-α after lipopolysaccharide treatment was diminished in BMDMs cultured with MSC-EVs (Supplemental Fig. 2, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). In the meantime, MSC-EVs reduced the level of M1 macrophage marker inducible nitric oxide synthase (iNOS) and enhanced the expression of M2 macrophage markers YM-1 and CD206 (Fig. 3A), indicating that MSC-EVs could polarize macrophages toward M2 phenotype. To examine the function of MSC-EV–cultured BMDMs, phagocytic activity of BMDMs was determined via flow cytometry analysis of engulfed Alexa Fluor 594–conjugated Escherichiacoli (Thermo Fisher Scientific). MSC-EVs significantly elevated the phagocytic activity of BMDMs in the presence of lipopolysaccharide compared with lipopolysaccharide alone (Fig. 3B). Then, we aimed to determine whether miR-27a-3p mediated the effect of MSC-EVs on macrophage polarization. Knockdown of miR-27a-3p in BMDMs via anti-miR-27a-3p lentiviral transduction blocked the effect of MSC-EVs on iNOS and CD206 expression as well as phagocytosis in the presence of lipopolysaccharide (Fig. 3C), indicating the essential role of miR-27a-3p in macrophage polarization.

Figure 3.
Figure 3.:
miR-27a-3p mediates the effect of mesenchymal stem cell–derived extracellular vesicles (MSC-EVs) on macrophage polarization in vitro. A, Bone marrow–derived macrophages (BMDMs) were cultured alone, with lipopolysaccharide (LPS) (100 ng/mL), or with MSC-EVs (100 µg/mL) plus LPS (100 ng/mL) for 24 hr. Then, BMDMs were harvested for quantitative real-time polymerase chain reaction (qRT-PCR) analysis to determine messenger RNA (mRNA) expression. M1 macrophage marker inducible nitric oxide synthase (iNOS) was reduced after culture with MSC-EVs. M2 macrophage markers YM-1 and CD206 were elevated after culture with MSC-EVs. B, BMDMs were cultured alone, with LPS (100 ng/mL), or with MSC-EVs (100 µg/mL) plus LPS (100 ng/mL) for 24 hr and then incubated with Alexa Fluor 594–conjugated Escherichia coli for 30 min. Phagocytosis was measured as fluorescence intensity per cell. C, BMDMs were transduced with lentiviral anti-miR-27a-3p or anti-miR control. Then, the cells were cultured with MSC-EVs (100 µg/mL) and simultaneously treated with LPS (100 ng/mL) for 24 hr. BMDMs were assayed for iNOS and CD206 mRNAs via qRT-PCR as well as phagocytosis via fluorescence intensity of engulfed E. coli. One-way analysis of variance with Bonferroni post hoc test (A), Kruskal-Wallis test with Dunn post hoc test (B), or Mann-Whitney U test (C) was used for the analysis. Data are presented as mean ± sd; n = 5–9. *p < 0.05, **p < 0.01, and ***p < 0.001. EV = extracellular vesicle.

Similar Effects Between MSC-EVs and MSCs in Alleviating Lipopolysaccharide-Induced Lung Injury and Macrophage Polarization

To compare the effects of MSC-EVs and MSCs in lipopolysaccharide-induced lung injury, mice were randomized to the following five treatment groups: control, lipopolysaccharide, lipopolysaccharide + MSCs IV (1 × 106 MSCs), lipopolysaccharide + EVs IV (50 µg), and lipopolysaccharide + EVs intratracheally (50 µg). MSCs IV, EVs IV, and EVs intratracheally were administered 0.5 hours after intratracheal lipopolysaccharide treatment. Intratracheal administration of lipopolysaccharide resulted in increased edema and cellularity in lung histology compared with control at 48 hours. Treatment with MSCs IV, EVs IV, and EVs intratracheally ameliorated the inflammatory cell infiltration and septal thickening (Fig. 4A). Increased pulmonary endothelial permeability is a hallmark of acute lung injury. The permeability was determined by total protein levels in the BAL of the above five groups at 48 hours after lipopolysaccharide treatment. Protein level was remarkably elevated in BAL after lipopolysaccharide treatment compared with that of the control. Compared with lipopolysaccharide-injured mice, treatment of mice with MSCs IV (p < 0.01), EVs IV (p < 0.001), and EVs intratracheally (p < 0.001) significantly reduced lung permeability by 26.0%, 38.2%, and 32.0%, respectively. No difference in permeability was observed among the three individual treatments (Fig. 4B). Treatment with MSCs IV, EVs IV, and EVs intratracheally also decreased the number of total cells (by 33.9%, 49.0%, and 40.4%, respectively) (Fig. 4C) and neutrophils in BAL (by 37.6%, 48.0%, and 43.5%, respectively) (Fig. 4D). Protective effects on lung permeability and cell infiltration were lost in injured mice treated with EVs IV or EVs intratracheally at 25 µg/mouse, although there was a trend toward reduced cell infiltration (Supplemental Fig. 3, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). Treatment with MSCs IV, EVs IV, and EVs intratracheally also significantly reduced proinflammatory cytokines including interleukin-1β, interleukin-6, and TNF-α in the BAL compared with the lipopolysaccharide group at 48 hours (Supplemental Fig. 4A, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). However, there was no statistical difference among the three groups.

Figure 4.
Figure 4.:
Both IV and intratracheal (IT) administration of mesenchymal stem cell–derived extracellular vesicles (MSC-EVs) alleviate lipopolysaccharide (LPS)-induced lung injury, elevate miR-27a-3p levels, and decrease nuclear factor kappa B subunit 1 (NFKB1) levels. Mice were divided into the following five groups: control, LPS, LPS + mesenchymal stem cells (MSCs) IV (1 × 106 MSCs), LPS + EVs IV (50 µg), and LPS + extracellular vesicles (EVs) IT (50 µg). A, Similar to the effects of MSCs, administration of EVs via both IV and IT dramatically improved lung injury as shown in histology. Lung sections were obtained at 48 hr after LPS insult and stained with hematoxylin and eosin. Both EVs IV and IT decreased protein concentrations (B), total cell counts (C), and neutrophil counts (D) in the bronchoalveolar lavage (BAL) harvested at 48 hr after LPS insult. Data are expressed as mean ± sd; n = 9. E, Alveolar macrophages were separated 48 hr after LPS insult and assayed for miR-27a-3p expression via quantitative real-time polymerase chain reaction. Results are presented relative to control group. Data are expressed as mean ± sd; n = 6. F, Alveolar macrophages were separated from BAL 48 hr after IT LPS insult and assayed for NFKB1 expression via Western blot analysis. Left, A representative blot was shown with two samples from EVs IV and EVs IT each. Right, The bands in the blots were densitometrically quantified. Data are expressed as mean ± sd; n = 6. One-way analysis of variance with Bonferroni post hoc test (B, C, and D) or Kruskal-Wallis test with Dunn post hoc test (E and F) was used for the analysis. *p < 0.05; **p < 0.01; and ***p < 0.001. PBS = phosphate-buffered saline.

To compare the effects of MSCs IV, EVs IV, and EVs intratracheally on alveolar macrophage polarization in mouse model of acute lung injury, BAL macrophages from the above five groups at 48 hours after lipopolysaccharide treatment were examined for markers for M1/M2 macrophages by qRT-PCR. There were some variations among MSCs IV, EVs IV, and EVs intratracheally in modulating macrophage phenotypes. Treatment with MSCs reduced the mRNA levels for M1 marker iNOS induced by lipopolysaccharide and elevated the expression of M2 marker arginase-1 but not interleukin-10. EVs IV mimicked the effects of MSCs IV but also increased the expression of interleukin-10. EVs intratracheally had similar effects on the expression of iNOS and interleukin-10 as EVs IV but did not alter the levels of arginase-1 (Supplemental Fig. 4B, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). Overall, these results support that administration of MSC-EVs via both intratracheally and IV could mimic the systemic effects of MSCs in acute lung injury and M2 macrophage polarization.

Transfer of miR-27a-3p From MSC-EVs to Alveolar Macrophages In Vivo and Repressing NFKB1 Expression

To determine whether MSC-EVs could transfer miR-27a-3p to macrophages in mouse model of lipopolysaccharide-induced lung injury, alveolar macrophages were harvested from different groups of mice as described in the previous paragraph (control, lipopolysaccharide, lipopolysaccharide + MSCs IV, lipopolysaccharide + EVs IV, and lipopolysaccharide + EVs intratracheally) at 48 hours after lipopolysaccharide treatment and examined for miR-27a-3p expression. miR-27a-3p levels in alveolar macrophages were significantly decreased after acute lung injury and elevated after treatment with MSCs IV, EVs IV, and EVs intratracheally (Fig. 4E). These results correlate with the results of our in vitro study and support the findings of a previous report that miR-27a expression was down-regulated in lipopolysaccharide-treated human macrophages (27). TargetScan and microrna.org computational methods predicted NFKB1, an essential part of lipopolysaccharide-induced NF-κB activation, as a miR-27a-3p target. Lipopolysaccharide treatment significantly increased the protein level of NFKB1 in BAL alveolar macrophages at 48 hours compared with control, whereas EVs IV and intratracheally abolished the effect of lipopolysaccharide on NFKB1 levels, suggesting miR-27a-3p might suppress NFKB1 expression (Fig. 4F).

Essential Role of miR-27a-3p in Mediating the Effects of MSC-EVs on Acute Lung Injury and M2 Macrophage Polarization

To establish that miR-27a-3p is responsible for modulating lung injury and M2 polarization induced by MSC-EVs, Lentivirus-mediated antisense miR-27a-3p (anti-miR-27a-3p) was transduced into MSCs to knockdown the expression of miR-27a-3p, whereas nonspecific anti-miR was introduced as control. Four days after transducing with a high multiplicity of infection (60) of Lentivirus, almost 100% of the MSCs expressed green fluorescent protein (GFP) fluorescence indicating an efficient transduction (data not shown). In mice with lipopolysaccharide-induced lung injury, the effects of intratracheally EVs from MSCs expressing anti-miR-27a-3p were compared with those of intratracheally EVs from MSCs expressing the anti-miR control. Anti-miR-27a-3p eliminated the beneficial effects of MSC-EVs and increased protein permeability, total cell count, and neutrophil infiltration in the BAL (35%, 80%, and 75%, respectively) at 48 hours (Fig. 5A). Anti-miR-27a-3p also blocked the effects of MSC-EVs on M2 polarization of alveolar macrophages in vivo by decreasing the expression of M2 markers CD206, FIZZ1, and YM-1 (Fig. 5B). In addition, anti-miR-27a-3p significantly increased NFKB1 protein levels compared with that of the control in Western blot analysis (Fig. 5C). Furthermore, qRT-PCR analysis showed that NFKB1 mRNA levels in BAL macrophages were elevated in the anti-miR-27a-3p group, further indicating that miR-27a-3p targets at NFKB1 (Fig. 5D).

Figure 5.
Figure 5.:
miR-27a-3p knockdown (anti-miR-27a-3p) in mesenchymal stem cells (MSCs) diminishes the therapeutic effect of MSCs–derived extracellular vesicles (MSC-EVs) by targeting nuclear factor kappa B subunit 1 (NFKB1). Thirty minutes after lipopolysaccharide (LPS) intratracheal (IT) treatment, mice were divided into two groups: LPS + IT extracellular vesicles (EVs) from MSCs transduced with anti-miR-27a-3p (50 µg) and LPS + IT EVs from MSCs transduced with anti-miR control (50 µg). Bronchoalveolar lavage (BAL) and alveolar macrophages were harvested 48 hr after LPS treatment. A, miR-27a-3p knockdown increased protein concentrations, total cell counts, and neutrophil counts in the BAL. B, Expression of markers for M2 (CD206, FIZZ1, and YM-1) in BAL macrophages was assayed by quantitative real-time polymerase chain reaction (qRT-PCR). C, NFKB1 protein levels in BAL macrophages were determined by Western blot analysis. Left, A representative blot was shown with two samples from each group. Right, The bands in the blots were densitometrically quantified. D, NFKB1 messenger RNA (mRNA) levels in BAL macrophages were determined by qRT-PCR. Mann-Whitney U test (A, B, and C) or Student t test (D) was used for analysis. Data are presented as mean ± sd; n = 4–8. *p < 0.05 and **p < 0.01.

Absence of Protective Effects of MSC-EVs on Lung Injury and M2 Polarization in Mice With miR-27a-3p Knockdown

To further corroborate the protective role of miR-27a-3p in lung injury conferred by MSC-EVs, lentiviral anti-miR-27a-3p was administered to mice via intratracheally 5 days before lipopolysaccharide challenge to knockdown miR-27a-3p in vivo. BAL macrophages were positive for lentiviral GFP at day 5 post transduction (Supplemental Fig. 5A, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). miR-27a-3p knockdown abolished the effects of MSC-EVs and elevated protein permeability, total cell count, and neutrophil infiltration in the BAL (58%, 56%, and 64%, respectively; p < 0.05) at 48 hours (Supplemental Fig. 5B, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). The effects of MSC-EVs on M2 macrophage polarization were also blocked by miR-27a-3p knockdown as evidenced by a reduction of CD206+ cells in BAL macrophages in flow cytometry assay (Supplemental Fig. 5C, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379), elevated expression of iNOS and interleukin-1β, and declined expression of arginase-1 (Supplemental Fig. 5D, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379).

miR-27a-3p Targeting at NFKB1

TargetScan and microrna.org predicted a putative miR-27a-3p binding site at the 3′UTR of mouse NFKB1. To determine whether NFKB1 is a direct target of miR-27a-3p, the putative miR-27a-3p binding sequence in the mouse NFKB1 3′UTR or a mutated NFKB1 3′UTR was cloned into downstream of a luciferase reporter (Supplemental Fig. 6A, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). Cotransfection of pGL3-NFKB1 3′UTR and pcDNA-miR-27a-3p into HEK293 cells resulted in a 30% decrease of luciferase activity compared with pGL3-NFKB1 3′UTR and pcDNA empty vector-transfected cells. Mutations in the 3′UTR miR-27a-3p binding site abolished the inhibitory effect of miR-27a-3p (Supplemental Fig. 6B, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379). These data demonstrate that miR-27a-3p binds to NFKB1 3′UTR directly and thereby attenuates its expression.

DISCUSSION

Studies from other groups in murine models of acute lung injury have demonstrated that administration of MSC-EVs recapitulated the beneficial effect of their parent MSCs. The present study reveals that MSC-EVs alleviate acute lung injury via transfer of miR-27a-3p to alveolar macrophages. This conclusion is substantiated by the following findings: 1) macrophages were able to uptake MSC-EVs in vitro and in vivo (Fig. 1); 2) miR-27a-3p was transferred from MSC-EVs to monocytes/macrophages in vitro (Fig. 2) and in vivo (Fig. 4E); 3) MSC-EVs inhibited macrophage activation and induced M2 macrophage polarization in vitro, effects that were blocked by anti-miR-27a-3p (Fig. 3); 4) administration of MSC-EVs via IV and intratracheally had similar effects in ameliorating acute lung injury (Fig. 4) and inducing M2 macrophage polarization (Supplemental Fig. 4, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379); 5) miR-27a-3p was at least partially responsible for the protective effect of MSC-EVs in vivo (Fig. 5; and Supplemental Fig. 5, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379); and 6) miR-27a-3p directly targeted at NFKB1 (Figs. 4F and 5, C and D; and Supplemental Fig. 6, Supplemental Digital Content 2, http://links.lww.com/CCM/F378; legend, Supplemental Digital Content 3, http://links.lww.com/CCM/F379).

In the present study, our results showed that miR-27a-3p expression in alveolar macrophages was significantly reduced in a mouse model of lipopolysaccharide-induced lung injury. This finding was consistent with a previous report that lipopolysaccharide down-regulated miR-27a expression in cultured macrophages (27). Former studies have postulated many miRNAs as biomarkers for acute lung injury. It remains to be determined whether miR-27a-3p in alveolar macrophages may serve as a marker for acute lung injury. Our results also presented that miR-27a-3p promoted M2 macrophage polarization. Previous studies using monocyte-derived macrophages from blood donors showed that miR-27a-3p was uniquely overexpressed in M2 macrophages (24). Also, miR-27a from EVs of alcohol-treated monocytes regulated differentiation and M2 polarization of naive monocytes (23,28). In addition to miR-27a-3p, there is a list of miRNAs that are involved in miRNA-mediated macrophage polarization. For example, miR-9, miR-155, and miR-125b skew macrophages toward M1 phenotype, whereas miR-223, miR-34a, and miR-146a induce M2 polarization via various target proteins (29).

In recent years, the literature supports that the effect of MSC-EVs in lung injury is mediated by modulating macrophage functions. Morrison et al (30) showed that MSCs favored an M2 macrophage phenotype, which is anti-inflammatory and highly phagocytic, through EV-mediated mitochondrial transfer. Adoptive transfer of macrophages pretreated with MSC-EVs alleviated lipopolysaccharide-induced acute lung injury. Phinney et al (31) documented that MSC-EVs transferred mitochondria from MSCs to macrophages, enhanced bioenergetics of acceptor macrophages, and reduced silica-induced lung inflammation. In a model of bronchopulmonary dysplasia, MSC-EVs improved lung function, reduced fibrosis, and alleviated pulmonary hypertension via modulation of lung macrophage phenotype (32). Our report demonstrates that MSC-EVs ameliorate acute lung injury via transfer of miRNA to alveolar macrophages and modulation of macrophage polarization.

There are several limitations/unexplained findings to this study worth noting. First, MSC-EV ultracentrifugation yielded 2.36 ± 0.63 × 108 particles/µg of protein and 417 ± 156 particles/cell at 24 hours post subculture. The dose of EVs administered to each animal (50 µg) was equivalent to 1.18 ± 0.32 × 1010 EV particles, which was similar to that of a report from Haga et al (33) on liver injury. On the other hand, the dose of administered MSCs (1 × 106 cells) would generate only 4.17 ± 1.56 × 108 EVs in 24 hours at culture condition. This disparity can be explained by the fact that MSCs may produce EVs with effective delivery of miRNAs to target cells at stress condition (34), although others may argue that the majority of MSCs do not survive over 24 hours in vivo (35). The disparity could also be explained by the fact that MSCs are able to exert their effects via paracrine factors and direct cell-to-cell interaction in addition to releasing MSC-EVs. Second, the functional difference between EV subtypes was not examined in acute lung injury model due to technical difficulty in separating subtypes. Based on our TEM analysis, MSC-EVs had a diameter of 50–150 nm in size. However, exosomes (50–150 nm) and microvesicles (100–1,000 nm) have an overlap in size. Previous studies have demonstrated that differences in size and buoyant density are not sufficient for a clear separation of exosomes and microvesicles (36). In addition, there are no consensus-specific markers for separating exosomes and microvesicles. Third, our analysis showed that the copy number for miR-27a-3p is relatively low with approximately 0.01 copy per MSC-EV particle (not shown in the results), which is similar to a reported average copy number of most abundant miRNAs from six EV sources (37). Based on the copy number, approximately 1.18 × 108 copies (50 µg × 2.36 × 108 particles/µg × 0.01) of miR-27a-3p were administered to the mice in the present study. This raised the question of whether miR-27a-3p from MSC-EVs may reach the physiologic level of approximately 100–1,000 copies of miRNA per cell estimated to be required to target miRNAs (38). However, recent studies have shown that miRNAs may also exert their effects through a novel mechanism by directly interacting with innate immune RNA sensors, such as Toll-like receptors (39,40).

CONCLUSIONS

In conclusion, miR-27a-3p is transferred from MSC-EVs to macrophages and induces polarization of M2 macrophages. Administration of MSC-EVs intratracheally and IV alleviates lipopolysaccharide-induced lung injury, which is associated with elevated miR-27a-3p levels, reduced NFKB1 levels, and induced M2 polarization of alveolar macrophages. miR-27a-3p is at least partially responsible for the effects of MSC-EVs in acute lung injury. The application of MSC-EVs or MSC-EVs with miR-27a-3p overexpression may be a viable option to treat acute lung injury.

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Keywords:

acute lung injury; mesenchymal stem (stromal) cells; acute respiratory distress syndrome; extracellular vesicles; macrophages; miR-27a-3p

Supplemental Digital Content

Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine and Wolters Kluwer Health, Inc.