T Cell-derived Exosomes Mediated Immunological Effects In Oral Lichen Planus

Oral lichen planus(OLP)is a chronic autoimmune disease mainly involving the oral mucosa.Although the pathogenesis of OLP has not been fully clarified,numerous evidences suggested that the development of OLP is closely associated with the T cellmediated antigen-specific mechanisms.Exosomes are extracellular vesicles with a size ranging from 50 to 150 nm,originated from the internal budding of the plasma membrane during endocytic internalization.Intraluminal vesicles released after fusion of multivesicular body(MVB)to plasma membrane are the so called exosomes.Once being engulfed by adjacent and distant cells,exosomes can modify the overall functions of target cells,being widely researched in many aspects of medical science such as autoimmunity,tumor,and infection.Recently,the biological capacity of T cell-derived exosomes(T-exos)in inflammatoty immune response is increasingly gaining attention.In the present study,we for the fisrt time investigated the immunological roles of T-exos in the pathogenesis of OLP.Based on the three parts of experiments,the research step by step revealed the following facts(1)the differential expression of MVB-related proteins in OLP T cells;(2)the effects of OLP T-exos on the proliferation and cytokine profile of T cells,as well as the influence on the apoptosis of keratinocytes;(3)the chemotactic function of MIP1α/β and the underlying mechanism.The results of this study demonstrated that T-exos exhibit multiple immunological effects on the development of OLP,including the regulation of cytokine secretion,the induction on keratinocytes apoptosis and the indirect induction on the migration of OLP CD8+T cells.Part 1.The expression of MVB-related proteins in OLP T cells and the clinical significanceObjective:The aim of the study was to detect the expression of MVB-related genes and/or proteins including CD63,CD81,Rab27A,Rab27B,Alix and Tsg101 in OLP lesional and peripheral T cells,and analyse the correlation between indicators’ mRNA levels and OLP severity.Materials and methods:Tissue samples and peripheral T cells were collected from OLP patients and healthy controls for RNA extraction.Gene levels of CD63,CD81,Rab27A,Rab27B,Alix and Tsg101 in tissues and peripheral T cells were measured using qRT-PCR.Co-localizations of CD3 with CD63,CD3 with Rab27A,as well as CD3 with Rab27B were determined by double-immunofluorescence staining.The severity of OLP was assessed by RAE score system;correlation between the scores and the gene levels of MVB-related proteins was analysed.Results:In comparison with healthy controls,the gene expression of CD63 and Rab27A was significantly upregulated in OLP,while Rab27B gene expression was significantly down-regulated.In OLP lesions,CD63 was densely infiltrated in the lamina propria and markedly co-localized with T cells,while few of Rab27A/B was colocalization with T cells.The mRNA levels of CD63,CD81 and Alix in OLP peripheral T cells were significantly higher than controls.Clinical correlation analysis showed that,in OLP lesions,the mRNA levels of CD63 was positively correlated with the corresponding RAE sores,while that of Rab27B was in a negative correlation with the disease severity.On the contrary,the gene level of CD63 in peripheral T cells was negatively correlated with the RAE sores,and so was the gene expression of Tsg101.Conclusions:These data suggested that an active biogenesis and secretion of exosomes may be present in OLP T cells,and exosomes released by OLP T cells might be implicated in the pathogenesis of OLP.Part 2.OLP T cell-derived exosomes regulated the cytokine secretion of T cells and induced the apoptosis of keratinocytesObjective:The present study was aimed to isolate and identify T-eoxs,and to explore the role of OLP T-exos in the production of cytokines by T cells and in the apoptosis of keratinocytes.Materials and methods:Peripheral T cells were separated from the venous bloods of OLP patients and healthy controls.Supernatants of human T cells were collected after 5～6 d cell cultures,and extracellular vesicles purified from the supernatants by filtration and ultracentrifugation were identified using NTA,WB and TEM.PKH-67labled T-exos and Dil-labled Jurkat T cells or keratinocytes were co-cultured,and the internalization of T-exos was observed by flow cytometry and/or confocal laser scanning microscope.After incubating Jurkat T cells with T-exos for 48h,expression profile of the 25 cytokines in the supernatants was determined by luminex assay.Proliferation of Jurkat T cells was assessed using CCK-8 kit.After co-culturing LPSpretreated human oral keratinocytes with T-exos for 24h,morphological alteration of keratinocytes was observed under optical microscope and cell apoptosis was assessed via flow cytometry.In addition,expression profile of the 25 cytokines in T-exos was determined by luminex assay.Results:The T cell-derived extracellular vesicles were homogeneously distributed with an average size of 118.7 ± 32.1 nm and a diameter peak at 117 nm,characterized by a classic cupstand-like morphology and high amounts of exosomal markers CD63 and CD9,indicating that these extracellular vesicles were exosomes.T-exos was incorporated by Jurkat cells and keratinocytes after incubating for 4 h and 24 h.OLP Texos inhibited the production of GM-CSF and IFN-γ by Jurkat T cells,and promoted the secretion of IL-10,IL-17A,and in particular MIP-1α and MIP-1β.Additionally,HC T-exos significantly weaken the proliferation of Jurkat T cells,while the OLP T-exos exhibited no inhibitory effect.The apoptotic rates of both HC T-exos and OLP T-exostreated keratinocytes were significantly increased in comparison with the PBS-treated group,and cells were altered obviously in morphology.In OLP T-exos,the expression level of IL-1β,IL-5 and IFN-γ was significantly reduced,the level of IL-7,10,12 and especially IL-17 was remarkably increased,while both OLP and HC T-exos contained high amounts of TNF-α.Conclusions:OLP T-exos may play an important role in the pathogenesis of OLP by regulating the cytokine production of T cells,inducing the apoptosis of keratinocytes and to some degree leading to the over-proliferation of lesional T cells.Part 3.MIP-1α/β induced the migration of OLP CD8+T cells via CCR1 and CCR5Objective:The present study was aimed to identify the expression of MIP-1α/β and their receptors CCR1,CCR3 and CCR5 in OLP,and to investigate the effect of MIP1α/β on OLP T cells migration and the underlying mechanism.Materials and methods:The protein levels of MIP-1α/β in tissues and plasma were detected by IHC and ELISA,respectively.The expression of CCR1,CCR3 and CCR5 on T cells and the CD4 or CD8 subtypes of the CCR1/3/5 positive cells were analysed by flow cytometry.Transwell assay was performed to investigate the chemotactic effects of MIP-1α/β on OLPT cells,with or without inhibition on CCR1 or CCR5.Cells migrating to the lower chambers were examinated by flow cytometry to determine the proportion of CD4 or CD8 positive cells.Following in vitro stimulation with different dosages of MIP-1α/β for 24 h,the mRNA levels of CCR1 and CCR5 in HC peripheral T cells were assessed by qRT-PCRResults:MIP-1α and MIP-1β were highly expressed in OLP tissues and plasm.CCR1 and CCR5 were markedly expressed on OLP peripheral T cells,and the majority of CCR1 and CCR5 positive T cells were CD8 positive.Besides,MIP-1α/β promoted the migration of OLP PBMCs,while inhibiting CCR1/5 significantly decreased the trafficking of OLP PBMCs,especially that of CD8+T cells.In addition,the expression of CCR1 and CCR5 in T cells were not significantly altered by MIP-1α/β.Conclusions:OLP T-exos induced MIP-1α/β may drive the trafficking of CD8+T cells after binding with CCR1/5 in OLP,which may be associated with the intra-epithelial infiltration of CD8+T cells in OLP.