The Study On The Role Of MiR-223-3p In Pathogenic Th17 Cells In EAU And Its Mechanisms

ObjectiveAutoimmune uveitis is an intraocular inflammatory disease characterized by autoreactive T cells-mediated damage in the uveal tissues and retina.The pathogenesis of experimental autoimmune uveitis is similar to that of human uveitis,and it is a good model for studying the pathogenesis of autoimmune uveitis and exploring new therapeutic avenue.Pathogenic T helper（Th）17 cells and the activation of IL-23/IL-17 signaling pathway are closely associated with the onset and development of autoimmune uveitis.Our previous results showed that miR-223-3p was significantly up-regulated in CD4⁺T cells from EAU mice.Recent studies have shown that aberrant expression of miR-223-3p can lead to a variety of autoimmune diseases,but the role of miR-223-3p in autoimmune uveitis as well as the mechanism are still unclear.In this study,using the EAU model induced by adoptive transfer of IRBP_1-20-specific T cells,we investigated the role of miR-223-3p in autoimmune uveitis and pathogenic Th17cells.In addition,we elucidated the signaling pathway of miR-223-3p regulating pathogenic Th17 cells.This study will help us to understand the pathogenesis of autoimmune uveitis and provide new ideas and theoretical basis for the clinical treatment of autoimmune uveitis.Methods1.We performed microarray analysis detecting miRNA expression profiles between naive and autoreactive CD4⁺T cells and selected the two miRNAs with the most up-regulated expression（miR-223-3p and miR-210-3p）for further study.q RT-PCR was used to verify the results of miRNA microarray.We analyzed the expression of miR-223-3p and miR-210-3p in CD4⁺T cells stimulated with increasing doses of IRBP_1-20.CD4⁺T cells isolated from immunized mice were cultured under different Th cell polarization conditions and q RT-PCR was used to detect the expression of miR-223-3p and miR-210-3p.We investigated the expression of miR-223-3p and miR-210-3p in Th17 cells at different times after EAU induction to determine the relationship between miR-223-3p,miR-210-3p and EAU development.2.ELISA was used to determine the effects of miR-223-3p and miR-210-3p on the production of IL-17 by Th17 cells.Because of the stronger effect of miR-223-3p on IL-17 production,we next focused on the role of miR-223-3p in the regulation of autoreactive Th17 cells.miRNA mimics and inhibitor were used to overexpress or knockdown miR-223-3p to assess the regulatory effect of miR-223-3p on the percentage of CD4⁺IL-17⁺T cells and the expression of pathogenic Th17 cell-related genes.IRBP_1-20-specific Th17 cells transfected with miR-223-3p inhibitor or control inhibitor were adoptively transferred into wild-type mice.Clinical and histological evaluations were performed to clarify the role of T cell-intrinsic miR-223-3p in the pathogenesis of EAU.T cells isolated from the adoptively transferred mice were co-cultured with IRBP_1-20 and APCs in vitro.ELISA was used to detect the secretion of IL-17 and IFN-γ.Flow cytometry was used to analyze the percentage of CD4⁺IL-17⁺T cell and CD4⁺IFN-γ⁺T cell.In addition,the percentage of CD4⁺Foxp3⁺T cell in splenic cells from the adoptively transferred mice was also analyzed by flow cytometry.DCs overexpressing miR-223-3p were co-cultured with autoreactive CD4⁺T cells and IRBP_1-20 under Th17-polarizing conditions,and IL-17 production was detected by ELISA.The expression of Th17-polarizing cytokines in miR-223-3p-overexpressing DCs was determined by q RT-PCR.3.The potential target genes of miR-223-3p were predicted by bioinformatics methods.Luciferase reporter assay,q RT-PCR and Western Blot were used for functional verification.A gain-and loss-of function assay was performed using lentivirus-mediated FOXO3 overexpression or FOXO3 si RNA to study the role of target gene in pathogenic Th17 cell response and the possible mechanisms.We searched the Gene Expression Omnibus database for raw data from gene expression profiles in patients with uveitis and healthy volunteers to explore the correlation between target gene and pathogenic Th17 cells.Results1.The microarray results showed that miR-223-3p and miR-210-3p were significantly up-regulated in CD4⁺T cells from EAU mice,which was further confirmed by q RT-PCR analysis.miR-223-3p and miR-210-3p were significantly upregulated in IRBP_1-20-specific Th17 cells.During the pathogenesis of EAU,the expression of miR-223-3p and miR-210-3p were significantly increased,and the expression changes of miR-223-3p were closely related to the severity of EAU disease.2.IL-17 production,Th17 cell percentages,and pathogenic Th17 cell-related gene（IL-17,RORγt,IL-22,IL-1R1,IL-23R and GM-CSF）expression were significantly increased in miR-223-3p-overexpressing T cells,while knockdown of miR-223-3p in T cells resulted in a significant decrease in IL-17 secretion,Th17 cell percentages,and pathogenic Th17-related gene expression.The mice that received Th17 cells transfected with miR-223-3p inhibitor developed less severe EAU compared with mice that received Th17 cells transfected with control inhibitor,mainly manifested by decreased clinical scores,ocular inflammatory cell infiltration and retinal structural disorders.ELISA and flow cytometric analysis showed that the production of IL-17and IFN-γand the percentage of Th17 and Th1 cell in T cells from the mice transferred with miR-223-3p inhibitor-transfected Th17 cells were significantly reduced as compared to the control mice.The percentage of Treg in splenic cells from mice given transfer of Th17 cells transfected with miR-223-3p inhibitor was significantly higher than that in the control group.Overexpression of miR-223-3p in DCs could promote the secretion of IL-17 in Th17 cells.Besides,miR-223-3p overexpression can increase the expression of IL-23 and IL-1βin DCs.3.The bioinformatics analysis showed that 3’UTR of FOXO3 contained a highly conserved putative binding element for the miR-223-3p.The luciferase report experiment showed that miR-223-3p could directly target FOXO3 and inhibit its expression.FOXO3 expression was significantly up-regulated when miR-223-3p was knock down in EL4 T cells.The gain-and loss-of function assay revealed that FOXO3could negatively regulate IL-17 production and pathogenic Th17 cell-related gene expression by partially inhibiting IL-23R expression.The results of GEO database analysis showed that in PBMCs of patients with uveitis,the expression of FOXO3 was down-regulated,while the expression of pathogenic Th17 cell-related genes was up-regulated,and there was a negative correlation between the expression of FOXO3 and pathogenic Th17 cell-related genes.In addition,a published clinical dataset revealed that the expression level of miR-223 was elevated in PBMCs of patients with active uveitis.ConclusionmiR-223-3p was significantly upregulated in IRBP_1-20-specific Th17 cells and positively regulated pathogenic Th17 cell responses.Using a T-cell transfer model of EAU,we demonstrated that knockdown of miR-223-3p decreased the pathogenicity of Th17 cells and ameliorated EAU.Mechanistic studies showed that miR-223-3p directly targeted FOXO3 and repressed its expression,resulting in increased IL-23R expression and pathogenic Th17 cell response.Our study reveals a previously unknown T cell-intrinsic miRNA pathway that promotes autoreactive Th17 cells and identifies the miR-223-3p-FOXO3-IL-23R axis as a potential therapeutic target in uveitis.