Mesenchymal Stem Cell-derived Extracellular Vesicles Alleviate Acute Lung Injury Via Transfer Of MiR-27a-3p

Part 1 The role of macrophage uptake of mesenchymal stem cells-derived extracellular vesiclesObjective:Adipose derived mesenchymal stem cells(ASC)are one of the most efficient producers of EVs at least in part by extracellular vesicles(EVs).In recent years,the literature supports that the paracrine mechanism of MSCs is mediated at least in part by extracellular vesicles(EVs),it had not been reported how EVs can gain entry into target cells such as macrophages,as immune cells in lung tissue.In this part,EVs were obtained by ultracentrifugation and further identified.In vitro and in vivo experiments showed that macrophages could ingested a certain amount of EVs.It further proves the role of EVs in the development of LPS induced lung injury.Method:MSCs were cultured in DMEM supplemented with EV-depleted serum obtained from ultracentrifugation.Protein concentration of EVs was determined by a BCA Protein Assay Kit.The morphology of EVs was analyzed by transmission electron microscopy.The size distribution and particle concentration of EVs were measured using nanoparticle tracking analysis or dynamic light scattering.The markers of MSCs and MSC-EVs were analyzed by Western blot.Isolated MSC-EVs were labeled with CD63.For in vitro assay in THP-1 cells were incubated with CD63-labeled EVs for 24 h at 37 ?,and were imaged by confocal laser scanning microscopy.For in vitro assay in BMDMs,BMDMs were incubated with CD63-labeled EVs for 6 h at 37 ?.Data were collected on a flow cytometer and analyzed using Flow Jo software.For in vivo assay,isolated MSC-EVs were labeled with Vybrant? Di D cell-labeling solution.Mice were treated with 5 mg/kg of LPS intratracheally(IT)and administered with labeled EVs(50 ?g/50?l)IT 30 minutes later.Bronchoalveolar lavage(BAL)was harvested 24 h later cells were analyzed via a BD LSRFortessa? flow cytometer.Results:Transmission electron microscopy(TEM)analysis revealed that MSC-EVs had a cup-shaped morphology with a diameter of 50-150 nm in size.Their size distribution as determined via DLS and NTA showed a peak at approximately 106 nm in diameter indicating that MSC-EVs were composed by both microvesicles and exosomes.The ratio of the number of EV particles to the protein content was 2.36±0.63×108 particles/?g,which has been suggested as an indicator for EV purity.In medium supplemented with EV-free serum,the concentration of MSC-EVs reached 417±156 particles per cell at 24 h 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).To study the uptake of MSC-EVs by monocytes/macrophages in vitro,Confocal microscopy results showed that THP-1 cells procured labeled MSC-EVs.More than 40% of BMDMs were positive for CD63(F4/80+CD63+)after 6 h.To determine EV uptake by macrophages in vivo,Flow cytometry results showed that more than 70% of BAL macrophages were F4/80+DID+ after 24 h.Conclusion:The extracellular vesicles secreted by ASCs can be isolated and identified.And the uptake of MSC-EVs by macrophages also can be measued.Part 2 The mechanism of mesenchymal stem cells-derived extracellular vesicles in acute lung injuryObjective:In the first part,it was found that the supernatant of ASCs can exceed to rich EVs,and these EVs can be uptake by macrophages within 6-24 hours.In this part,we explore the mechanism of uptake of EVs by macrophages can affect the release of inflammatory factors in vitro and in vivo,and reduce acute lung injury.Method:To determine the effect of MSC-EVs on macrophage polarization,BMDMs were cultured with or without MSC-EVs and treated with LPS for 24 h.Then,total RNA was extracted from BMDMs and subjected to qRT-PCR analysis.To examine the function of MSC-EV-cultured DMEMs,phagocytic activity of DMEMs was determined via flow cytometry analysis using Alexa Fluor 594 conjugated E.coli.To compare the effects of MSC-EVs and MSCs in LPS-induced lung injury,mice were randomized to the following 5 treatment groups: control,LPS,LPS+MSCs IV(1×106 MSCs),LPS+EVs IV(50 ?g),and LPS+EVs IT(50 ?g).MSCs IV,EVs IV,and EVs IT were administered 0.5 h after IT LPS treatment.Lung histology,total protein levels,cell infiltration including total cells and neutrophils were compared among the 5 groups.BAL macrophages from the above 5 groups at 48 h after LPS treatment were examined for markers for M1/M2 macrophages by qRT-PCR,NFKB1 protein expression was test by Western blot.The expression percentage of CD206 on M2 macrophage surface was analyzed by flow cytometry.The BAL was analyzed with proinflammatory cytokines by Cytometric Bead Array(CBA).Results:The increase in mRNA levels of proinflammatory IL-6,IL-1?,and TNF-? after LPS treatment was diminished in BMDMs cultured with MSC-EVs(p<0.05).In the meantime,MSC-EVs reduced the level of M1 macrophage marker i NOS and enhanced the expression of M2 macrophage markers YM-1 and CD206(p<0.05).MSC-EVs significantly elevated the fluorescence intensity of BMDMs in the presence of LPS compared with LPS alone(p<0.05).Treatment with MSCs IV,EVs IV,and EVs IT ameliorated the inflammatory cell infiltration and septal thickening.Protein level was remarkably elevated in BAL after LPS treatment compared with control.Compared with LPS-injured mice,treatment of mice with MSCs IV(p<0.05),EVs IV(p<0.05),and EVs IT(p<0.05)significantly reduced lung permeability by 26.0%,38.2%,and 32.0% respectively.No difference was observed among the three individual treatment.Treatment with MSCs IV,EVs IV,and EVs IT also decreased the number of total cells(by 33.9%,49.0%,and 40.4% respectively),neutrophils(by 37.6%,48.0%,and 43.5% respectively).Protective effects on lung permeability and cell infiltration were lost in injured mice treated with EVs IT at 25 ?g/mouse.Treatment with MSCs IV,EVs IV,and EVs IT also reduced proinflammatory cytokines including IL-1?,IL-6 and TNF-?(p<0.05)in the BAL compared with the LPS group at 48 h.However,there was no statistical difference among the three groups.There were some variations among MSCs IV,EVs IV and EVs IT in modulating macrophage phenotypes.LPS treatment significantly increased the protein level of NFKB1 in BAL alveolar macrophages at 48 h compared with control,while EVs IV and IT abolished the effect of LPS on NFKB1 levels(p<0.05).Overall,these results support that administration of MSC-EVs via both IT and IV could mimic the systemic effects of MSCs in acute lung injury and M2 macrophage polarization.Conclusion:EVs derived from MSCs can lead to M2 macrophage polarization,reduce the release of inflammatory factors,inhibit the recruitment of neutrophils,and alleviate acute lung injury.Part 3 Mesenchymal stem cell-derived extracellular vesicles alleviate acute lung injury via transfer of miR-27a-3p to macrophagesObjective:The second part of the study had showed that MSCs-EVs can lead to M2 macrophage polarization.miRNAs can been transferred between cells as a way of intercellular communication.In this part,we aimed to determine whether micro RNA mediated the effect of MSC-EVs on macrophage polarization.Method:To explore the transfer of miRNAs between MSCs and monocytes/macrophages in vitro,human THP-1 monocytes were cultured alone(LPS)or cocultured with human MSCs(LPS+MSCs)via Transwell in the presence of LPS.micro RNA expression in THP-1 cells was compared between the two groups after microarray analysis.Human and murine miR-27a-3p sequences share 100% homology.THP-1 or BMDMs were cultured in the presence or absence of MSC-EVs or MSCs with or without LPS treatment,levels of miR-27a-3p in THP-1 cells and BMDMs were validated by qRTPCR.Knockdown of miR-27a-3p in BMDMs via anti-miR-27a-3p lentiviral transduction.The effect of MSC-EVs on IL-1?,IL-6,i NOS,and CD206 expression as well as phagocytosis were analyzed.5 groups in vivo at 48 h after LPS treatment were examined for miR-27a-3p expression.Target Scan and microrna.org computational methods predicted the target of miR-27a-3p.Lentivirus-mediated antisense miR-27a-3p(anti-miR-27a-3p)was transduced into MSCs to knockdown the expression of miR-27a-3p,the effects of IT EVs from MSCs expressing anti-miR-27a-3p were compared with those of IT EVs from MSCs expressing the anti-miR control in vivo.NFKB1 levels were compared with control in Western blot analysis.qRT-PCR analysis NFKB1 mRNA levels in BAL macrophages were also compared between two groups.To further corroborate the protective role of miR-27a-3p in lung injury conferred by MSC-EVs,lentiviral anti-miR-27a-3p were administered to mice via IT 5 days before LPS challenge to knock-down anti-miR-27a-3p in vivo.Protein permeability,total cell count,and neutrophil infiltration in the BALwere measured.The effects of MSC-EVs on M2 macrophage polarization was compared in BAL macrophages in flow cytometry assay and qRT-PCR.Results:miR-27a-3p was the second most highly expressed miRNA in the list and has been reported to participate M2 macrophage polarization,elevated levels of miR-27a-3p in THP-1 cells and BMDMs with coculture of MSCs or EVs were validated by qRT-PCR(p<0.05).Human and murine miR-27a-3p sequences share 100% homology.pri-miR-27 a levels in BMDMs were unchanged when incubated with MSC-EVs,indicating a direct transfer of miR-27a-3p rather than a transcriptional upregulation.LPS decreased miR-27a-3p expression in BMDMs via transcriptional mechanism as reflected by reduced pri-miR-27 a levels(p<0.05).anti-miR-27a-3p blocked the effect of MSC-EVs on IL-1?,IL-6,i NOS,and CD206 expression as well as phagocytosis induced by LPS in BMDMs.Lentiviral anti-miR-27a-3p were administered to mice via IT,miR-27a-3p knockdown abolished the effects of MSC-EVs(p<0.05).miR-27a-3p levels in alveolar macrophages were significantly decreased after acute lung injury and elevated after treatment with MSCs,EVs IV,and EVs IT(p<0.05).Target Scan and microrna.org computational methods predicted NFKB1,an essential part of LPSinduced NF-?B activation,as a miR-27a-3p target.anti-miR-27a-3p transduced MSC-EVs were eliminated the beneficial effects on M2 polarization of alveolar macrophages(p<0.05),and significantly increased NFKB1 levels(p<0.05)compared with control in Western blot analysis.qRT-PCR analysis showed that NFKB1 mRNA levels were elevated in anti-miR-27a-3p group(p<0.05),luciferase reporter assay demonstrated that miR-27a-3p binds to NFKB1 3?UTR directly and thereby attenuates its expression.Conclusion:miR-27a-3p was transferred from MSC-EVs to monocytes/macrophages in vitro and in vivo.MSC-EVs induced M2 macrophage polarization,effects that were blocked by anti-miR-27a-3p.miR-27a-3p was at least partially responsible for the protective effect of MSC-EVs in vivo.miR-27a-3p directly targeted at NFKB1.