Role Of Circulating Exosomes In Regulating T-cell Mediated Inflammatory Response In Oral Lichen Planus

Oral lichen planus(OLP)is a chronic immune inflammatory disease in oral mucosa whose immunopathogenesis may involve in antigen presentation,T cell activation,proliferation,apoptosis,and migration as well as keratinocytes apoptosis.The pathogenesis of OLP remains unclear at present,but T cells were confirmed closely associated with the immune response of OLP.Exosomes,as small homogeneous membrane-bound vesicles,are formed by fuse of multivesicular bodies and plasma membranes.They can be found in various body fluids,including peripheral blood,urine,milk,etc.Exosomes contain a great variety of bioactive molecules,such as proteins and nucleic acids,which could be taken up by receptor cells,and regulate cell-to-cell signaling transmission and then influence cell biological functions.It is reported that exosomes have emerged as potent stimulators of immune responses and play important roles in multiple immune and inflammatory diseases.Exosomes are reported to regulate the activation,proliferation,migration and apoptosis of T cells,leading to their dysfunction and biological alteration.However,the function and mechanism of exosomes in OLP remain unclear.The current study mainly aimed to investigate roles of circulating exosomes in regulating T-cell mediated inflammatory response in OLP patients,which might provide novel biomarkers for diagnosis and treatment of OLP.This study was divided into four parts as follows:Part 1.Comparison of circulating exosomes by three different methodsObjective: The standardized purification techniques to isolate exosomes with high quality are still deficient at present.This study was to compare the circulating exosomes isolated by three methods to select a superior one for further research.Materials and methods: Peripheral blood was collected from eleven healthy individuals.Exosomes were isolated by membrane-based affinity binding method,differential ultracentrifugation,and solvent precipitation-based method.Morphology was evaluated by transmission electron microscope;particle size distribution was measured by laser diffraction instrument and q Nanosight;exosomal biomarkers of CD9 and CD63 were detected by flow cytometry and western blot.Moreover,total proteins and RNA concentration were evaluated by BCA and Nanodrop 2000,respectively.Results: Membranous microvesicles with a diameter of 100 nm were confirmed in plasma by the three isolating methods,showing a typical cup-shaped structure.Flow cytometry and western blot identified exosomal biomarkers CD9 and CD63,indicating the successful isolation of exosomes by the three methods.The size distribution showed that particles by membrane-based affinity binding method were most uniform with one peak of the curve.The amount of proteins and RNA concentration showed no significant difference between membrane-based affinity method and ultracentrifugation.Exosomes isolated by solvent precipitation kits showed a significant higher amount of protein yield due to plasma albumin contamination,and the RNA concentration was significantly lower than the other two methods.Conclusions: All the three methods were confirmed successfully isolating circulating exosomes.Moreover,exosomes by differential ultracentrifugation and membranebased affinity binding method were purer and more appropriate for further analysis.Part 2.Circulating exosomes regulate T-cell mediated inflammatory response in oral lichen planusObjective: Exosomes were able to modulate the immune response and regulate T cell activation,proliferation,migration,and apoptosis,leading to T cell dysfunction.This study was aimed to investigate the effects of circulating exosomes extracted from OLP patients on T cells.Materials and methods:Plasma-derived exosomes were purified from OLP patients and normal individuals.T cells were observed under a confocal laser scanning microscope after co-cultivated with PKH67 labeled exosomes for 0 h,12 h,24 h,and 48 h.The effects of exosomes on T cells were analyzed by several functional assays,including proliferation,apoptosis,and migration.Production of interleukin(IL)-2,IL-4,IL-10,and interferon(IFN)-? was measured by enzyme-linked immunosorbent assay.Results: PKH67 labeled exosomes were taken up by T cells in a time-and dosedependent manner.Exosomes failed to activate T cells.Compared to the control group,circulating exosomes from erosive OLP significantly enhanced T cell proliferation,attenuated the apoptosis and promoted the migration ability,but no significant difference was found in the nonerosive OLP group.In addition,ELISA showed that circulating exosomes from erosive OLP contributed to the IFN-? secretion of T cells,but decreased the IL-4 expression.Conclusions:Our findings revealed that circulating exosomes from OLP patients were involved in the biological functions of T cells and might promote the progression of OLP by regulating T-cell mediated inflammatory response.Part 3.Differentially circulating exosomal micro RNAs expression profiling in oral lichen planusObjective: The aberrant expression of exosomal miRNAs in many diseases were significant for cell-to-cell signaling transmission.The aim of the present study was to identify circulating exosomal miRNAs expression profiles in OLP patients,and analyze their relationship with the clinical characteristics.Materials and methods:Plasma exosomes were isolated from OLP patients and healthy individuals.Total RNA was isolated from the plasma exosomes,plasma,and peripheral blood T cells.The RAE scoring system was used to evaluate the severity of OLP.Deregulated exosomal miRNAs associated with inflammatory response and autoimmunity were identified by mi Script? miRNA PCR Array,and the results were confirmed by q RT-PCR.The relationship between exosomal miRNAs and RAE scores was analyzed.Finally,bioinformatics analysis was used to predict the target genes and pathways of the differentially expressed exosomal miR-34a-5p.Results: Compared to control group and nonerosive OLP group,circulating exosomal miR-34a-5p and miR-130b-3p were upregulated,while miR-301b-3p was downregulated in erosive OLP patients.In addition,compared to control individuals,expression of miR-34a-5p and miR-130b-3p were increased,but miR-301b-3p was significantly decreased in nonerosive OLP.Exosomal miR-34a-5p was positively correlated with the severity of OLP.Moreover,expression of miRNA-34a-5p from the plasma and peripheral blood T cells of OLP patients was significantly increased.There was a positive correlation between the expression of miRNA-34a-5p in T cells and RAE scores.The bioinformatics analysis revealed that target genes of miR-34a-5p mainly involved in regulation of gene expression,cell communication,signaling,and metabolic process through PI3K/AKT signaling pathway.Conclusions: The expression of miR-34a-5p in circulating exosomes and peripheral blood T cells could be a potential biomarker for evaluating the severity of OLP.Part 4.MiR-34a-5p affects the biological functions of T cells through targeting PI3K/AKT/UCN2 signaling pathwayObjective: MiR-34a-5p was down-regulated in a variety of tumor cells and considered as an important tumor suppressor.However,its regulatory role in immune cells remains unclear.This study was to explore whether miR-34a-5p was involved in the regulation of T cells and the potential regulatory mechanism.Materials and methods:T cells were transfected with lentivirus miR-34a-5p mimics,miR-34a-5p negative control(NC),and miR-34a-5p inhibitors,then the proliferation and apoptosis of T cells were detected through CCK8 assay and flow cytometry.The prediction results of target genes were verified by q RT-PCR.The 3'UTR binding site of miR-34a-5p to the target gene was identified via the miRanda online database and double luciferase reporter gene system.The expression level of associated signaling pathway protein molecules was analyzed through western blot.Results: MiR-34a-5p mimics,miR-34a-5pNC,and miR-34a-5p inhibitors lentivirus were proved successfully transfected into T cells.Compared to the NC group,an increased proliferation and decreased apoptosis of T cells were found in mimics group,however,there was a reverse result in inhibitors group.The q RT-PCR and double luciferase reporter assay showed that miR-34a-5p could target UCN2 3'UTR in T cells.In addition,the expression of PI3 K,p-AKT,and UCN2 was negative associated with expression of miR-34a-5p in T cells.Conclusions: MiR-34a-5p could regulate T cells through targeting PI3K/AKT/UCN2 signaling pathway,indicating its involvement in OLP progression.