Investigation Into The Expression And Mechanism Of CD161 In Autoimmune Uveitis

Background:Uveitis is an inflammation of the iris,ciliary body,choroidal tissue,vitreous body retina and retinal blood vessels.The etiology of uveitis is complicated,but uveitis caused by autoimmune disorder is the most common,while autoimmune uveitis is more common for Behcet's disease and Vogt-Koyanagi-Harada syndrome.Autoimmune uveitis has a long course and is prone to recurrent attacks and difficult to treat,which seriously affects patients'visual function and quality of life.In recent years,the immunopathogenesis of Behcet's disease has been extensively studied,and found that a variety of antigen or autoantigen,innate immune cells,and adaptive immune cells and cytokines are involved in the process of uveitis.CD4⁺T cells,including Th1,Th17,Th22 and Treg cells and its related cytokines in the pathogenesis of uveitis plays a key role in the process.Therefore,it is of great significance to explore the mechanism of inflammatory response and adaptive immune system regulation in patients with uveitis from the perspective of immunology and molecular biology to find the therapeutic target of uveitis.CD161 molecule is a homomeric dimer C-type lectin.This surface molecule was originally identified as the human homologue of NKRP1 glycoprotein expressed on rodent NK cells,demonstrating 46-47%homology with its rodent counterparts.Recent studies have confirmed that CD161 is also expressed in human T cells.In peripheral blood lymphocytes,CD161 is not only expressed in CD4⁺TCR??⁺T cells,but also in CD8⁺TCR??⁺T cells,CD4^-CD8^-TCR??⁺T cells and CD4^-CD8^-TCR??⁺T cells.CD161^intCD4⁺and CD161^highCD8⁺T cells have also been isolated from fetal umbilical cord blood?UCB?,where they exhibit naive phenotypes.In the presence of Th17 polarized cytokine culture,IL-17 production was limited to the CD161⁺CD4⁺CD45RA⁺part,suggesting that Th17 cells were derived from progenitor cells of CD161⁺CD4⁺T cells.Moreover,CD161 may be involved in cellular immune response as a cellular marker produced by IL-17.A large number of studies have shown that CD161 can activate induced T cell amplification,polymorphism of CD161,recognition of other receptors on CD161⁺T cells and a series of functions,making CD161 a potential therapeutic target for a variety of diseases.The cAMP response element regulatory factor??CREM??regulation is a member of the transcription factor superfamily.More than 20 CREM isoforms in humans are achieved through differential splicing processes,transcription factors,promoters,and promoter codons.CREM expression is tightly regulated through tissue-cell and development-specific mechanisms.Thus,it is not surprising that CREM-mediated gene transcription has been extensively studied in cell systems that display selective expression of single CREM variants,such as male germ cells,cells of the adrenal and pituitary glands,and T lymphocytes.Human T lymphocytes predominantly express the CREM?isoform,and recent studies have shown that CREM?activates the transcription of certain cytokine genes in CD4⁺T cells and also affects the epigenetic modification and transcriptional regulation of cytokines in the IL-17 family.CREM?is considered to be a central contributor to tissue-specific gene regulation in immune cells.Therefore,it is speculated that CD161 and CREM may be involved in the pathogenesis of autoimmune uveitis,and there may be a mutual regulatory relationship between the two and play a role in the pathogenesis of autoimmune uveitis.Objective:The purpose of this study was to detect the expression level of CD161 in peripheral blood of T cells in patients with active uveitis.The mechanism of action of CD4⁺CD161⁺T cells was explored,and the possible regulatory relationship between CREM gene and CD161 molecule was further investigated.Methods:1.Exploring the distribution and expression of CD161 molecule on T cell surface in peripheral blood of patients with active uveitis.In this study,BD and VKH,the most common patients with autoimmune uveitis,were selected as subjects.Flow cytometry was used to detect the distribution and expression of CD161 before and after treatment in patients with acute BD and VKH,and normal subjects without autoimmune diseases were used as controls.Luminex was used to detect the expression of IL-17A and other cytokines in serum of the control group and patients with acute BD and VKH before and after treatment.2.Exploring the effect of cyclosporine on CD161 molecule.Cyclosporine was co-cultured with peripheral blood mononuclear cells from patients with active uveitis and control group in vitro.The expression of CD161 in T cells of BD and VKH patients and control group before and after cyclosporine was detected by flow cytometry.Luminex was used to detect the expression of IL-17A and other cytokines in the cell culture supernatant of BD and VKH patients and control group before and after cyclosporine treatment.3.Detecting the expression of CREM?in patients with acute autoimmune uveitis.CD4⁺T cells in peripheral blood of patients with active uveitis and control group were isolated by immunomagnetic beads,and the transcriptional and protein expressions of CREM??CD161?ROR?t and IL-17A in peripheral blood CD4⁺T cells of acute BD and VKH patients were investigated by RT-qPCR and Western blot.4.Detecting the CREM?regulated expression of CD161 in patients with acute autoimmune uveitis.CREM?siRNA was transfected into CD4⁺T cells of peripheral blood of patients with acute BD and VKH by small interference RNA technology to inhibit the expression level of CREM protein.mRNA and protein expressions of CD161?ROR?t and IL-17A were detected by RT-qPCR and Western blot.Results:1.CD161 molecules were distributed in CD3⁺,CD3⁺CD4⁺and CD3⁺CD8⁺T cells,and tche proportion of CD161⁺CD3⁺and CD161⁺CD3⁺CD4⁺T cells in peripheral blood of BD and VKH patients in the active phase was significantly higher than that in the control group.The level of CD161⁺Th17 cells in peripheral blood of patients with active BD and VKH was significantly higher than that of healthy control group and post-treatment group.The levels of CD4⁺CD161⁺and CD4⁺CD161^-T cells secreting IL-17A in the peripheral blood of the control group and the patients with acute BD and VKH before and after treatment were further analyzed,and the results showed that the levels of CD4⁺CD161⁺T cells secreting IL-17A were significantly higher than those of CD4⁺CD161^-T cells.The expression of IL-17A and other cytokines in the serum of the control group and the patients with acute BD and VKH before and after treatment was detected,and the results showed that the IL-6,IL-17A,IFN-?and TNF-?levels of the patients with acute BD and VKH were higher than those of the healthy control group and the patients after treatment.2.When cyclosporine was added in vitro,the expression level of CD161 and the proportion of Th17 cells in BD and VKH patients at the active stage could be significantly down-regulated.At the same time,the expression level of CD161 and the proportion of Th17 cells in the control group were significantly down-regulated,so its effect was non-specific.The expression of cytokines such as IL-1?,IL-2,IL-12p70,IL-17A,TNF-?,IFN-?and GM-CSF in the cell culture supernatant of the control group and the active BD and VKH patients also showed that cyclosporine could non-specifically reduce the expression levels of these cytokines.3.The mRNA transcription and protein expression levels of CREM??CD161?ROR?t and IL-17A in peripheral blood CD4⁺T cells of patients with acute BD and VKH were significantly higher than those in control group and post-treatment group.4.After transfection of CREM?siRNA into CD4⁺T cells of patients with acute phase BD and VKH,CREM?expression was inhibited,along with reduced transcription and protein expression levels of CD161?ROR?t and IL-17A.Conclusion:CD161 molecules in CD4⁺and CD8⁺T cells were distributed,and active in patients with autoimmune uveitis high expression of peripheral blood CD4⁺T cells,and CD4⁺CD161⁺T cells were associated with IL-17A secretion,and CREM acts as a transcription factor mediating the expression regulation of CD161?ROR?t and IL-17A as a molecular mechanism involved in autoimmune development.While controlling clinical inflammation,cyclosporine can inhibit the frequency and cytokine expression of CD161 and Th17 cells,suggesting that CD161 or CREM may be a new target for the treatment of autoimmune uveitis.