Bone Marrow Mesenchymal Stem Cell-derived Exosomes Regulate Ly6C Monocyte Subsets In Vitro

Myocardial infarction(MI)remains a leading cause of morbidity and mortality worldwide with a large financial burden on the economy.Post-MI,with the loss of functional cardiomyocytes,the heart undergoes adverse remodeling with progressive thinning of the infarcted wall,cardiac dilatation and loss of contractile function,all of which can lead to heart failure.The rapid death of cardiac myocytes in the ischemic heart induce inflammatory signals recruit monocytes/macrophages to the infarct zone,these leukocytes degrade extracellular matrix constituents and macromolecules released by injured cells and aid clearance of dead cardiac myocytes and their debris.After 5 d after MI,monocytes/macrophages enhance the synthesis of type I collagen by myofibroblasts to strengthen the infarct and protects it against rupture.Monocytes/macrophages are widely involved in the inflammatory response,defense and tissue remodeling processes.Monocytes are produced in the bone marrow in the steady-state from hematopoietic precursors.Monocytes produced in the bone marrow enter the blood via CCR2.In the mouse,circulating monocytes are phenotypically and functionally heterogeneous and can be separated according to Ly6C expression.Between 50%and 60%of mouse monocytes in the steady-state belong to the Ly-6Chigh CCR2hish CX3CR1low subset.These inflammatory or classical monocytes secretes proinflammatory cytokines such as IL-6 and accumulate preferentially in inflanImatory sites where they give rise to macrophages.The remaining Ly-6Clow CCR2low CX3CR1high subset,sometimes referred to as nonclassical,anti-inflammatory subpopulation that expresses high levels of anti-inflammatory cytokines such as IL-10,plays a positive role in inflammation repair and scar repair.Inflammatory Ly-6Chigh monocytes/macrophages accumulate early,whereas reparative Ly-6Clow accumulate later in the inflammatory diseases of the heart,liver and kidney,Evidence shows that monocyte subsets do not arise from separate progenitors,but rather convert from the Ly-6Chigh to the Ly-6Clow subset.However,the fate of Ly-6Clow monocyte in bone marrow and their function in heart disease requies further study.Mesenchymal stem cells(MSCs)are stromal cells with cardiac regenerative properties that have been demonstrated to reverse cardiac dysfunction and enhance angiogenesis in damaged heart tissue.Furthermore,MSCs switched from infiltration of pro-inflammatory to anti-inflammatory macrophages for alleviating inflammation and augmenting cardiac regeneration.In a sepsis model,MSCs can secrete certain growth factors to increase the percentage of reparative M2 macrophages and improve organ function.Increasing data indicate that MSCs may create an optimal microenvironment for reducing in ammation and promote tissue repair through a paracrine mechanism,and exosomes play an important role in this process.Exosomes are small membranous vesicles that contain bioactive molecules,including protein,messenger RNAs(mRNAs)and microRNAs(miRNAs),which can be transferred between cells and thus modulate cellular activities and reprogram the phenotype in recipient cellsExosomal miRNAs have been shown to play an important role in myocardial infarction.In the regulation of inflammation,exosomal miRNAs can promote the differentiation of Treg cells and transform M1 macrophages into M2 macrophages.However,it remains unclear how MSCs resolve chronic inflammation and whether they may function by accommodating Ly6C monocyte subsets.We investigated whether exosome-derived lipopolysaccharide(LPS)preconditioned MSCs have a regulatory effect on the bone marrow-derived Ly6C monocyte subsets in inflammation resolution and the role of miRNAs in exosomes played in this process.Methods:1.Primary human bone marrow-derived mesenchymal stem cells(BMSCs)were isolated by gradient centrifugation and identified by cell morphology,surface marker and osteogenic differentiation.BMSCs were cultured with serum-free medium for 48 h,and exosome were purified from cultured-conditional medium by using ExoQuick reagent.Nanoparticle Trafficking Analysis(NTA)was used to detect the diameters and concentration,ultrastructure of exosomes was determined by transmission electron microscope.The surface markers(CD9,CD63,CD81)were identified by Amnis ImageStream Mark II.Furthermore,the expression of other proteins(Alix,HSP70,Flotillin-1)were identified by Western Blot.2.Using different stimulations treated BMSCs to mimic inflammatory stress,the exosomes were collected and co-cultured with monocytes to select the ideal stimulating factor in vitro.The difference of the size and concentration of exosomes was analyzed by Nanosight.The monocytes were isolated from the tibia and femur of the mice and sorted by CD11b magnetic beads.Cells were co-cultured with exosomes for 3 days.The proportion of Ly6C monocytes/macrophages was measured by flow cytometry,the relative expression of chemokines was detected by RT-qPCR,ELISA investigated inflammatory cytokines in cell supernatant.3.The miRNAs in unstimulated exosomes and LPS-stimulated exosomes were sequenced to find miRNAs with significant difference.According to the related references,changed miRNA was selected to study further.The miRNA target genes were predicted by TargetScan,miRDB,miRTarBase,and miRWalk,and common genes were used for subsequent GO functional enrichment analysis and KEGG pathway enrichment analysis;After treating with exosomes,Western Blotting measured expression levels of TLR4 in monocytes to initially analyze the potential mechanism of exosomal effects on monocytes.Results:1.The primary culture of BMSCs presented as long spindle-shaped fibrocyte-like adherent cells and gathered in a swirling pattern and grew rapidly.They have osteogenic and adipogenic differentiation capacity.BMSCs expressing antigens were determined by Flow Cytometry,BMSCs were positive for CD29,CD44,and CD90,CD73,CD105,CD166,but negative for CD31,CD34,CD45.To extract the BMSCs-derived exosomes,the culture medium of BMSCs was collected and precipitated.NTA showed that the diameters mode of exosomes was 69.2 ± 3.0 nm and the concentration of the particles was 6.67×109±1.36×108 particles/mL,most of the particles are in the exosomes diameter range(30-100nm).Transmission electron microscope(TEM)revealed particles were double-layer membrane structure and diameters about 100 nm,and the surface transmembrane proteins CD9,CD63 and CD 81 were detected by flow cytometry with a fluorescence microscope.Other exosomal protein Alix,HSP70 and Flotillin-1 were detectable with western blotting.2.The exosomes produced by LPS stimulated BMSCs significantly changed the ratio of Ly6C monocyte subsets.After treated with LPS,NTA results showed that the exosomes concentrations increased from 7.75×1011±2.06×107 particies/L to 3.58×1012±6.12×107 particles/L from BMSCs were treated with 500ng/?L LPS for 24 hours,but the diameter did not change significantly(82.40±3.70 vs 91.90±15.90)nm.Most of the nanoparticles were in the range of exosome diameter(30-100 nm).Flow cytometry showed that the proportion of Ly6Chigh monocytes were significantly increased with Ly6Clow monocytes were decreased under treatment with exosome-derived lipopolysaccharide(LPS)preconditioned BMSCs(LPS-exo)compared with control group.Both RT-qPCR and ELISA showed that LPS-exo increased the expression levels of pro-inflammatory cytokines IL-1?,IL-6,chemokine CCL2 and CX3CL1,and anti-inflammatory cytokines IL-10 and TGF?.3.Combining the sequencing results and related literature,there are seversal highly differentially expressed miRNAs in LPS-exo,which are involved in a variety of biological functions and signaling pathways,including inflammation-related PI3K-Akt-mTOR signaling pathway.Westen blot detected the changes of related pathway proteins and found that LPS-exo could stimulate the expression of TLR4,indicating that TLR4 is involved in the role of exosomes in monocyte subpopulations and miRNA is indispensable in Ly6C monocytes heterogeneity.Conclusion:1.We successful isolated human BMSCs and harvested BMSCs derived exosomes for the following experiment.2.BMSCs have potential to secrete more exosomes under the stimulus of LPS and could significantly change the proportion of bone marrow derived Ly6C monocyte subsets in vitro.Exosomes can obviously increase the number of Ly6Chigh monocytes and reduce Ly6Clow monocytes.(convert from the Ly-6Chigh to the Ly-6Clow subset)It also increased the expression levels of proinflammatory cytokines IL-1?,IL-6 and anti-inflammatory cytokines IL-10,TGF?,which suggest that exosomes have effect both on Ly6Chigh monocytes and Ly6Clow monocytes,and the increased relative expression of chemokines CCL2 and CX3CL1 indicates that exosomes enhance the the mobilization and recruitment of monocytes from the bone marrow into the bloodstream.3.MiRNAs was selectively chosen into the exosomes under LPS treatment and increased the expression of the gene TLR4 involved in the transfirmed process through the activation of the signaling pathway,exosomes regulated the proportion of bone marrow derived-Ly6C monocyte subsets and activated inflammatory pathways to promote the release of inflammatory cytokines.Therefore,these results suggest a novel role for BMSCs-derived exosomes in regulating bone marrow derived-Ly6C monocyte subsets into pro-inflammatory phenotype,the first time demonstrated the mechanism of transformation,which is mainly through exosomal miRNA activate TLR4 in monocytes through the corresponding signaling pathway.This research provides a novel immune regulation role of MSCs paracrine effect on monocytes as part of inflammatory microenvironment and a useful therapeutic strategy for inflammatory diseases.