Exosomes Derived From Stem Cells From Apical Papilla Inhibit Th17 Differentiation Through Hsa-piR-15254 In The Treatment Of Sjogren's Syndrome

Objective:Sjogren's syndrome(SS)is a chronic autoimmune disease characterized by progressive lymphocyte infiltration and decreased secretory function of salivary glands.There are many features of SS,such as dry mouth,swelling of salivary glands and lacrimal glands,and more severe cases can be accompanied by systemic multi-system immune damage,which seriously affects the quality of life of patients.Lymphocyte infiltration and dysfunction of T cells are thought to be pivotal in the pathogenesis and progression of SS.T helper 17(Th17)cells are the highly proinflammatory T cell subset,which may play an important role in SS pathogenesis.Mesenchymal stem cells(MSCs),which have self-renewal capacities,multiple-lineage differentiation potentials and a wide range of immunoregulatory properties,provide a new therapeutic direction for SS.Exosomes derived from MSCs(MSC-Exo)are a type of nano-scale membranous vesicles secreted by MSCs,which has similar immunomodulatory properties to MSCs and shows efficacy against SS.Stem cells from apical papilla(SCAP)are a group of adult stem cells derived from neural crest,which are isolated from the apical papilla of immature permanent teeth.Great advantages presented by SCAP include a high ex vivo expansion capacity,ample cellular sources,accessibility without iatrogenic trauma and especially the strong ability of regulating T cell transformation.Whether exosomes derived from SCAP(SCAP-Exo)can treat SS remains unknown and the underlying mechanism is unclear.Non-codingRNA is considered to be the main substance in MSC-Exo,which exerts its function.PIWI-interactingRNA(piRNA)is a newly discovered endogenous non-coding smallRNA,with a capacity of regulating cell phenotype and function by targeting binding to the 3'UTR region of mRNA and inducing its degradation,which plays an important role in the pathogenesis of autoimmune diseases and T cell differentiation.In this experiment,SCAP-Exo were infused into the tail vein of SS mice to observe its effects on saliva secretion,lymphocyte infiltration in submandibular gland and the proportion of Th17 cells in peripheral blood and spleen of SS mice.In addition,we explored the regulatory effect of SCAP-Exo and its piRNA on the differentiation of Th17 in vitro,providing a basis for the application of exosomes derived from dental stem cells in SS.Methods:1.SCAP was primary isolated with enzymatic digestion.Flow cytometry,osteogenic and adipogenic differentiation experiments were used to identify SCAP.SCAP-Exo was extracted by ultracentrifugation;nanoparticle tracking analysis(NTA)and transmission electron microscopy(TEM)were used to identify their morphology and size.Western blot was performed to detect the expression of exosomal specific marker proteins Alix,CD9,CD63 and the specific protein Calnexin of the endoplasmic reticulum.2.NOD/Ltj mice were used as SS animal model.SS mice were identified by detecting the level of saliva secretion and HE staining to observe the degree of lymphocyte infiltration in submandibular gland.Then SCAP-Exo was infused into SS mice through tail vein to detect their saliva secretion level,the proportion of Th17cells in submandibular gland,spleen and peripheral blood,the expression level of IL-17A in serum,and the degree of lymphocyte infiltration in submandibular gland.3.CD4~+T cells were isolated from peripheral blood by immunomagnetic beads,and the induction system of CD4~+T cells to Th17 was constructed.After adding different concentrations of SCAP-Exo(20,40?g/m L),the proportion of Th17 cells was detected by flow cytometry,the expression of IL-17A in the supernatant was detected by ELISA,and the mRNA expression of Th17-related genes IL-17A,IL-21 and ROR?t were detected by qRT-PCR.4.The piRNAs in SCAP-Exo and BMMSC-Exo were sequenced with high throughput technology,and the piRNAs expression profiles were analyzed,using bone marrow mesenchymal stem cells-derived exosomes(BMMSC-Exo)as the control group.In order to screen the piRNAs which were related to Th17 cells differentiation,we performed prediction and bioinformatics analysis of the target genes of the top 40 piRNAs expression in SCAP-Exo.Through data analysis,we selected hsa-piR-15254 as the target piRNA,IL-6R as its candidate target gene.5.The endocytosis of PKH67 fluorescence labeled SCAP-Exo by CD4~+T cells was observed,and the expression of hsa-piR-15254 was detected by qRT-PCR after CD4~+T cells were treated with SCAP-Exo.The targeted binding of hsa-piR-15254 to IL-6R was detected by luciferase report experiment.Then we constructed hsa-piR-15254mimics and hsa-piR-15254 inhibitor,and added them to the induction system of differentiation of CD4~+T cells to Th17.The proportion of Th17 cells was detected by flow cytometry,the expression level of IL-17A in cell supernatant was detected by ELISA,and the mRNA expression levels of IL-6R,IL-17A,IL-21 and ROR?t were detected by qRT-PCR.6.SPSS 21.0 statistical software was used to analyze the data.The pairwise comparison of data was analyzed by variance analysis,and the submandibular gland pathological scores were graded by rank-sum test.P<0.05 was considered statistically significant.Results:1.Flow cytometry revealed that SCAP expressed MSCs markers(CD73,CD90,and CD105)but failed to express hematopoietic stem cells markers.After 4 weeks of SCAP osteogenic induction,mineralized nodules formed and alizarin red S staining was positive,while lipid droplets and oil red O staining were positive after 4 weeks of adipogenic induction.TEM imaging showed that SCAP-Exo was cup-shaped and NTA indicated that the diameters of SCAP-Exo ranged approximately from 30 to 150nm.Western blot analysis showed that SCAP-Exo had immunoreactivity with specific antibodies against CD9,CD63,and Alix but failed to express Calnexin.2.NOD/Ltj mice had the pathological characteristics of SS.HE staining showed that there were obvious lymphocyte infiltrating masses in their submandibular glands,and the level of saliva secretion was significantly lower than that of healthy mice(P<0.01).After SCAP-Exo was infused into the tail vein,the saliva secretion level of SS mice was significantly improved;HE staining showed that the infiltrating mass of submandibular gland lymphocytes decreased even disappeared.Immunofluorescence staining revealed that CD4 and IL-17A double-positive cells were significantly reduced.Flow cytometry showed that the proportion of Th17 cells in their spleen and peripheral blood was decreased,the level of IL-17A in serum was decreased by ELISA,and the differences were statistically significant(P<0.01).3.The purity of CD4~+T cells isolated from peripheral blood by immunomagnetic beads method was more than 90%.SCAP-Exo at the concentration of 20?g/m L and40?g/m L could inhibit the differentiation rate of CD4~+T cells to Th17,and the concentration of IL-17A in the supernatant and the mRNA expression of Th17-related genes IL-17A,IL-21 and ROR?t were significantly down-regulated,and the inhibitory effect of 40?g/m L SCAP-Exo was more obvious(P<0.05).4.High-throughput sequencing showed that piRNAs were abundant in SCAP-Exo,and a total of 593 piRNAs expressions were identified.The target genes of the top 40piRNAs expressed significantly enriched to the Th17 cells differentiation signal pathway.Hsa-piR-15254 was highly expressed in SCAP-Exo and may target IL-6R,a key signal in the differentiation pathway of Th17 cells.Therefore,hsa-piR-15254 was selected as the target piRNA to study the mechanism of SCAP-Exo regulating the differentiation of CD4~+T cells to Th17 cells.5.Fluorescence-labeled SCAP-Exo could be endocytosis by CD4~+T cells and up-regulate the expression of hsa-piR-15254 in CD4~+T cells.Luciferase reporter assay confirmed the targeted binding of hsa-piR-15254 to IL-6R.The high expression of hsa-piR-15254 in SCAP-Exo could inhibit the differentiation of CD4~+T cells to Th17 by targeting combined with IL-6R.The concentration of IL-17A in the supernatant and the mRNA expression of Th17 related genes IL-17A,IL-21 and ROR?t were significantly down-regulated(P<0.05).Conclusions:SCAP-Exo improved the saliva secretion,reduced submandibular gland lymphocyte infiltration and down-regulated the proportion of Th17/CD4~+T cells in peripheral blood and spleen and the expression level of IL-17A in serum of SS mice.In vitro experiments,SCAP-Exo could be endocytosed by CD4~+T cells,transported hsa-piR-15254 to regulate the expression of IL-6R,and inhibited the differentiation of CD4~+T cells to Th17 cells and the expression of Th17-related genes and proteins,thereby exerting their immunoregulatory properties.