Serum Exosomes As Biomarkers For Diagnosis Of Acute Ischemic Stroke And Their Role In Post-ischemic Inflammatory Response

Part 1 Characterization of mouse serum exosomal small RNAs content: The origins and their roles in modulating inflammatory response Objective: To explore the contents,the origins and biological signifcance of serum exosomes small RNAs under normal conditions.Methods: Serum exosomes were purified by Exoquick precipitation and then identified by morphology and molecular phenotype using transmission electron microscopy,nanoparticle tracking analysis(NTA)and western blot,respectively.Moreover,we profled the small RNAs content of mouse serum exosomes using small RNAseq and identifed known mi RNAs and predicted novel miRNAs.Based on the obtained mouse serum exosomal miRNAs profile and existing literature regarding cell and/or tissue specifc miRNAs,we proposed the possible sources of the mouse serum exosomes.The prediction of targets for the top 20 exosomal miRNAs and following molecular and cellular functional analyses of these predicted targets were carried out by using the Ingenuity Pathway Analysis(IPA)software.Finally,the uptake of SYTO-labeled exosomes by mouse macrophage cell line RAW264.7 was observed using immunofluorescence microscopy and the effect of mouse serum exosomes(mSEs)on the phenotype of RAW 264.7 cells were detected by qRT-PCR.Results: In the present study,particles obtained from C57BL/6 mouse serum by ExoQuick precipitation conformed to the major characteristics of exosomes.We found that fragments of transfer RNAs(tRNAs)and miRNAs were the two predominant exosomal RNA species by using small RNAs sequencing,accounting for approximately 60% and 10% of mapped reads,respectively.Moreover,466 known and 11 novel miRNAs were identifed from two independent experiments,among which the five most abundant mi RNAs(miR-486a-5p,miR-22-3p,miR-16-5p,miR-10b-5p and miR-27b-3p)accounted for approximately 60% of all the aligned miRNA sequences.As inferred from the identities of the well known cell-or tissue-specifc miRNAs,mSEs were primarily released by RBCs,liver and intestinal cells.Bioinformatics analysis revealed over half of the top 20 miRNAs by abundance were involved in inflammatory responses and further in vitro experiments demonstrated that mSEs potently primed macrophages towards the M2 phenotype.Conclusion: The results of this study provide a reference for future biomarker studies and extrapolating their origins and also suggest the roles of mSEs in maintaining internal homeostasis under normal conditions.Part 2 Acute ischemia increased the levels of a panel of brain specific miRNAs in mouse serum exosomes: a comprehensive study based on small RNA-seq Objective: to explore the potential of serum exosomal miRNAs as diagnostic and prognostic biomarkers of ischemic stroke.Methods: Transient middle cerebral occlusion was induced in C57BL/6 mice.Serum exosomes were purified by Exoquick precipitation.Effects of ischemia on the physical features of serum exosomes,miRNAs content of serum exosomes and brain tissues and exosome depleted serum were detected by nanoparticle-tracking assay(NTA),small RNAseq and qRT-PCR.Results: Ischemic brain injury increased serum exosomes concentration and changed the exosomal miRNAs profile greatly.Among the differentially modulated miRNAs,a panel of upregulated brain specific or enriched miRNAs were identified,notably,most of them were undetectable or very low in sham group.Temporal analysis of the four brain specific mi RNAs(miR-9-5p,miR-124-3p,miR-129-5p and mi R-344d-3p)revealed the levels of these mi RNAs increased after the onset of ischemia and returned to normal conditions after recovery.The dynamics of these mi RNAs in ischemic hemisphere was complementary to that in serum exosomes,moreover,they were almost lost in the ischemic core but maintained a relatively high level in border 1 day and 3 days after ischemia.In addition,except miR-124-3p,whose change was significantly higher in both serum exosomes and exosomes depleted serum,the level of the other three brain specific miRNAs only increased in serum exosomes.Conclusion: This study revealed the appearance and/or increase of a panel of brain specific miRNAs in the serum exosomes after acute ischemic brain injury,and the relationship between these miRNAs and BBB damage and brain injury.This study will promote diagnostic and prognostic biomarker screening of cerebral ischemia injury,and provide an experimental basis for understanding the biological significance of the appearance of these mi RNAs in serum exosomes.Part 3 Serum exosomes convey the inflammatory signaling Objective: To explore the effect of LPS on the physical characteristics and composition of serum exosomes and the role of serum exosomes in convey the inflammatory signaling.Methods: Serum exosomes were purified by Exoquick precipitation.Effects of LPS treatment on the physical features and RNAs concentration of mouse serum exosomes were detected by nanoparticle-tracking assay(NTA)and Agilent Bioanalyzer 2100.The effects of LPS treatment on the content of serum exosomes miRNAs,the expression of inflammatory cytokines in plasma exosomes and peripheral blood lymphocytes of mice,and protein expression levels of IL-1β and IL6 in serum exosomes were detected by small RNAseq,qRT-PCR and Elisa kit and the biological functions of differentially expressed miRNAs were analyzed using IPA software.Moreover,the uptake of SYTO-labeled exosomes by cells in vitro and in vivo were observed using immunofluorescence microscopy and laser scanning confocal microscopy,respectively.qRT-PCR was used to detected the effects of LPS-treated mice serum exosomes on the expression of inflammatory cytokines in RAW 264.7 and BV2 cells and in peripheral blood lymphocytes,brain and liver of C57BL/6 mice.Results: Compared to the PBS-treated control,LPS treatment significantly increased the concentration and RNAs content of serum exosomes.Interestingly,serum exosomes could be taken up effectively by cells in vitro and in vivo.More importantly,we found that LPS-treated mice serum exosomes group identified more miRNAs by using small RNA sequencing,and LPS treatment also significantly altered the miRNA expression profile of mouse serum exosomes.IPA software analysis found that some of these differentially expressed miRNAs play important roles in disease-related inflammation.Moreover,LPS treatment can simultaneously modulate the mRNAs expression of inflammatory cytokines in plasma exosomes and peripheral blood lymphocytes and both of them have similar inflammatory cytokine mRNAs expression profiles.In addition,LPS treatment increased the protein expression levels of the pro-inflammatory cytokines IL-1β and IL6 in serum exosomes.LPS-treated mice serum exosomes can promote the expression of inflammatory cytokines in RAW 264.7 and BV2 cells and in peripheral blood lymphocytes,brain tissues and liver tissues of normal mice.Conclusion: Serum exosomes could convey LPS-induced inflammatory signals in vitro and in vivo by changing their miRNAs,mRNAs and proteins content.These findings will help to further understand the role of exosomes in the transmission of signals and the pathophysiology of inflammation associated with disorders and provide an experimental basis for further explore the pathogenesis of the disease.Part 4 Serum exosomes from ischemic brain injury mice promoted microglia activation Objective: To explore the biological function of differentially expressed miRNAs in serum exosomes from ischemic brain injury mice and the role of serum exosomes in promoting microglial activation.Methods: The IPA software was used to analyze the biological functions of 62 differentially expressed miRNAs between the sham and MCAO mice serum exosomes obtained in the part 2 and 18 miRNAs which are within the top 100 as well as differentially expressed.Transient middle cerebral occlusion was induced in C57BL/6 mice,the uptake of SYTO-labeled exosomes by BV2 cells and microglia in MCAO mouse were observed using immunofluorescence microscopy and laser scanning confocal microscopy,respectively.In addition,the effects of serum exosomes from MCAO mice on the expression of inflammatory cytokines in BV2 cells was detected by qRT-PCR.Results: IPA software analysis showed that 62 differentially expressed mi RNAs were mainly associated with the network functions related to organismal injury and abnormalities,immune diseases and inflammatory diseases.In addition,the top 20 miRNAs in each group of serum exosomes from Sham and MCAO mice accounted for more than 70% of the total miRNAs,even the 100 most abundant mi RNAs accounted for more than 97% of the total miRNAs.18 miRNAs which are within the top 100 as well as differentially expressed were mainly associated with the network functions related to organismal injury and abnormalities.Further studies showed that serum exosomes could be taken up effectively by BV2 cells and microglia in MCAO mouse.MCAO mice serum exosomes could convey the inflammatory signaling to promote the expression of inflammatory cytokines in BV2 cells.Conclusion: Differentially expressed miRNAs in MCAO mouse serum exosomes are highly correlated with inflammatory responses and induced microglial activation.These studies would contribute to the treatment of stroke.