Effect Of MiRNA-155 On Melanocytes By Inhibiting The CD8~+ T Cells Via Upregulating Treg Cells In Vitiligo

BackgroundVitiligo is a common acquired disease characterized by white spots on the skin Accumulating evidence suggests that vitiligo is caused by the loss and degradation of epidermal melanocytes.Several theories have been proposed for the development of this disease,including autoimmunity,cytotoxic metabolites,neural and genetic causes and induction of oxidative stress.These factors have been suggested to explain the mechanisms of the melanocyte degradation,although the exact pathogenesis remains unknown.Recent studies have shown that vitiligo is an autoimmune response targeting melanocytes.Cytotoxic CD8+T cells can specifically recognise melanocytes,which can in turn be isolated from the lesions of vitiligo subjects,and it has been found that the intensity of CD8+ T cell responses is related to disease severity..Therefore,CD8+T cells may play a critical role during the processes of melanocyte loss and degradation.While,Treg cells were significantly decreased in active generalized vitiligo.In addition,the functional defect of Treg cells was involved in the pathogenesis of vitiligo.Therefore,it was considered that the decrease in the number of natural Treg cells may cause the activation of CD8+T cells,which can in turn damage the structure of melanocytes and lead to immune function disorders.These studies indicate that CD8+T cells and Treg cells play an important role in the pathogenesis of vitiligomicroRNAs(miRNAs)are small conserved non-coding RNA molecules,which have been found to play key roles in normal cellular processes and are considered to be key regulators of post-transcriptional gene in mammals.These miRNAs typically target one or more messenger RNAs(mRNAs)and degrade mRNAs or inhibit translation of target genes by binding to complementary sequences of the 3'-untranslated region(3'-UTR)of the target gene after transcription.miRNA-155(m iR-155)is a non-coding transcript derived from activated lymphocytes and high expression in monocytes/macrophages.It mainly inhibits the translation process by incompletely complementary pairing with the target gene,and down-regulates the expression level of the target genes.Previous studies have proposed that miRNA-155 is a crucial regulator in the process of inflammation and immunity.It plays an important role not only in the development and maturation of immune cells,but also in regulation of immune response.A recent study demonstrated that miR-155 was dysregulated in patients with vitiligo,suggesting that miR-155 may be involved in the pathogenesis of vitiligo,but the mechanisms by which miR-155 regulates the development of vitiligo remain unclear.In addition,miR-155 can increase the differentiation of Treg cells by activating the transcription of forkhead box P3(Foxp3),a marker of Treg cells.Therefore,we hypothesized that miRNA-155 can inhibit the proliferation of CD8+ T cells via upregulating Treg cells to protect melanocytes in vitiligo through the immune pathway.Objective1.To study the differential expression of miRNA-155 in peripheral blood T cells of vitiligo patients and healthy donors;2.To explore the regulation of miRNA-155 on the differentiation and function of Treg cells in patients with vitiligo;3.To study the effect of miRNA-155 on the activation and apoptosis of CD8+ T cells via regulating Treg cells in patients with vitiligo;4.To investigate the effect of miRNA-155 on the melanocytes via regulating Treg cells in patients with vitiligo.Materials and methods1.Purification of CD3+ T cells from vitiligo patients and healthy donors.Peripheral blood mononuclear cells were obtained from nine patients with non-segmental vitiligo and six healthy donors by Ficoll-Hypaque density gradient centrifugation.CD3+ T cells were sorted by CD3 antibody and the purity of CD3+ T cells was evaluated using flow cytometry.2.miR-155 expression in T cells of the patients with vitiligo and healthy donor was detected by Quantitative real-time PCR.3.Purification of naive T and CD8+ T cells from vitiligo patients.Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque density gradient centrifugation.CD3+CD4+CD45RA+T cells(naive T)were sorted by CD3,CD4,and CD45RA antibodies and CD3+ CD8+ T cells(CD8+ T cells)were sorted by CD3 and CD8 antibodies.The purity of the two selected T cells were evaluated using flow cytometry.4.The differentiation of naive T cells to Treg cells.The isolated naive T cells were plated into plates coated with anti-CD3 and anti-CD28.The cells were treated with IL-2(100 U/mL),TGF-?(10 ng/ml),and retinoic acid(10 nM).Following 4 days of stimulation,flow cytometry was used to determine the purity of CD4+CD25+FoxP3+Treg cells5.Nucleofection.A human T Cell Nucleofector(?)Kit and a nucleofector device were used for nucleofection.Initially,1×107 Treg cells were resuspended in 100 ?l of nucleofector(?)solution.Subsequently,100 pM oligonucleotides(including pre-miR-155,pre-miR-ctrl,anti-miR-155 or anti-miR-ctrl)were added to the solution,and mixed gently.The mixtures were gently transferred to the electric rotor and placed in the nucleofector device.Treg cells were nucleofected in the X-01 program.Lastly,the cells were transferred to a 12-well plate,prepared with 1.5 ml human T cell nucleofector medium,and incubated at 37? in a 5%CO2 incubator.The transcription level of miRNA-155 was detected by qRT-PCR after 24 hours.6.The effect of miRNA-155 on the differentiation and function of Treg cells.3 days after nucleofection,the ratio of CD4+CD25+FoxP3+ Treg cells in each group was detected by flow cytometry.The expression of Foxp3 was detected by qRT-PCR and Western blot and the expression levels of IL-10 and TGF-?1 were detected by qRT-PCR and ELISA.7.Cell culture.Primary melanocytes were isolated from a patient with vitiligo by suction blister and cultured in Hu 16 medium at 37? in a 5%CO2 incubator.The cell density was 5×105/ml in the culture flasks,and the base factors were added to the medium to remove keratinocytes and fibroblasts on the third day.The cells were subjected to logarithmic growth for 2 to 3 passages for subsequent experiments,and L-Dopa staining was used to identify melanocytes.8.Experimental groups:A:melanocyte;B:melanocytes+CD8+T cells;C:melanocytes+Treg cells;D:melanocytes+Treg cells + anti-miR-ctrl+CD8+T cells;E:melanocytes+Treg cells+miRNA-155 antagonist(anti-miR-155)+CD8+T cells;F:melanocytes+Treg cells+pre-miR-ctrl+CD8+T cells;G:melanocytes+Treg cell+miRNA-155 agonist(pre-miR-155)+CD8+T cells;Subsequently,the melanocytes were seeded in 6-well plates at a density of 1×105 cells/ml,placed in an incubator for 4 h and inoculated with CD8+ T cells,Treg cells,pre-miR-155 or anti-miR-155 at a ratio of 1:5:59.The effect of miRNA-155 on the activation and apoptosis of CD8+T cells via regulating Treg cells.After 3 days of cell incubation,the ratio of CD4 CD8+and CD69+CD137+cells was evaluated with FACSCalibur flow cytometry.The induction of CD8+T cells apoptosis was detected using the Annexin V-FITC/PI apoptosis detection kit following the manufacturer's instructions.The number of apoptotic cells was quantified by flow cytometry10.The effect of miRNA-155 on the apoptosis of melanocytes via regulating Treg cells.After 3 days of cell incubation,the apoptosis of melanocytes was detected using the Annexin V-FITC/PI apoptosis detection kit following the manufacturer's instructions.The number of apoptotic cells was quantified by flow cytometryResults1.The purity of CD3+T cells of the patients with vitiligo and the healthy donors was higer than 99%respectively.Also the purity of CD3+CD4+CD45RA+T cells of the patients with vitiligo was higher than 99%,the purity of sorted CD3+CD8+T cells was 95.32%.The purity of CD4+CD25+Foxp3+Treg cells was 93.15%;2.The level of miR-155 in T cells of the patient with vitiligo was downregulated compared with that in the healthy donor3?Treatment with anti-miR-155 significantly downregulated the level of miR-155 in Treg cells,while pre-miR-155 exhibited the opposite effect,as compared with the corresponding control groups4.miR-155 increased the percentage of Treg cells.We further examined the effects of miR-155 on the differentiation of Treg cells using flow cytometry.The results revealed that anti-miR-155 caused a significant decrease in the percentage of Treg cells,while pre-miR-155 exhibited the opposite effects in the cell culture medium.Similarly,anti-miR-155 inhibited significantly the gene and protein levels of Foxp3,while pre-miR-155 exhibited the opposite effects.It indicated that miR-155 positively regulate Treg cell differentiation.5.In order to investigate the effects of miR-155 on the function of Treg cells,the mRNA levels of IL-10 and TGF-?1 were assessed in T cells and the extracellular secretions of these cytokines in the culture medium were examined.The results indicated that pre-miR-155 significantly increased IL-10 and TGF-?1 mRNA expression levels,while anti-miR-155 markedly downregulated the levels of these cytokines.The extracellular secretions of IL-10 and TGF-?1 were significantly increased in the pre-miR-155 group,while they were downregulated in the anti-miR-155 group.It indicated that miR-155 can positively regulate Treg cell function.6.The effects of miR-155 on CD8+ T cells were subsequently evaluated using flow cytometry.The results showed that pre-miR-155 significantly reduced the percentage of CD8 T cells,while anti-miR-155 showed the opposite effect.CD69 and CD137 play important roles in CD8+ T cell activation.The results showed that there was no significant difference in the expression level of CD69 between different groups,while the expression level of CD137 was down-regulated after pre-miR-155 treatment.7.In addition,after 3 days co-culture,the results of flow cytometry showed that Treg cells induced apoptosis of CD8+ T cells,which was further enhanced by pre-miR-155 treatment,and apoptosis of CD8+ T cells was significantly increased.It indicated that miR-155 could promote apoptosis of CD8+ T cells via regulating Treg cells.8.After 3 days co-culture,the flow cytometry results showed that CD8+ T cells could induce apoptosis in melanocytes,which was partly reversed by the function of Treg cells.In addition,CD8+ T cell-induced apoptosis was markedly inhibited by the application of pre-miR-155.Conclusion1.The level of miR-155 in T cells of the patient with vitiligo was downregulated.2.miR-155 positively regulated the differentiation of Treg cells,increased the percentage of Treg cells,up-regulated the transcription of Foxp3 and positively regulated the function of Treg cells.3.miR-155 decreased the percentage of activated CD8 T cells by promoting apoptosis of CD8 T cells induced via regulating Treg cells.4.Treg cells were able to inhibit the apoptosis of melanocytes induced by CD8 T cells.miR-155 was able to enhance the inhibitory effect via regulating Treg and protect melanocytes.