Teff And Treg Cell Population Dynamics In Vitiligo Vulgaris And The Mechanism Of Melanocytes Destruction

Vitiligo is an acquired depigmentation disorder characterized by the appearance of circumscribed white macules in the skin due to the chronic, progressive loss of functional melanocytes (MC) in the epidermis. The impact of vitiligo on patients’ psychological well-being and social interactions could not be underestimated. Currently, the exact aetiology of vitiligo remains obscure, and the acceptable permanent cure is lack. We therefore explore the pathogenesis of vitiligo and hope to find the appropriate therapeutic targets, which may aid in the future development of effective immunotherapy for vitiligo patients.Accumulated data from the literature have provided substantial evidence to support autoimmune pathogenesis of a vitiligo vulgaris (W). High frequencies of Melan-A specific CD8+T cells were detected in the peripheral blood and lesional skin of W patients, which seemed to correlate with the extent and the activity of the disease. Moreover, a statistically significant increase of additional autoimmune diseases (AID) in W patients and their family members has been observed. So far, however, the pathomechanism underlying the loss of immune tolerance in W is still unknown. Regulatory T cell (Treg) plays an essential role in maintaining immunologic homeostasis and preventing autoimmunity. The deficiency and dysfunction of Tregs were involved in various human AID. Preliminary results from other group have shown that Treg were functionally defective in patients with VV. Th17and Th22are two newly discovered inflammatory effector subsets of CD4+T cells, which are involved in a variety of AID pathogenesis. Their roles, however, in the pathogenesis of VV are still not clear. We therefore hypothesized that a quantitive and functional failure of Treg might be fail to control the hyper-activation of effector T (Teff) subsets, which leaded to the development and progress of W and associated AID and allergic disorders (ALD).We demonstrated here that the prevalence of AID and ALD, including autoimmune thyroiditis, chronic urticaria, bronchial asthma and systemic lupus erythematosus (SLE) in patients with VV were significantly higher compared with those in the normal population, suggesting that a hyper-activation of immune response presented in VV patients. Furthermore, we detected an imbalance between Teff and Treg population, and found that the hyper-activation of Teff subsets and the depletion of Treg were correlated with VV flare. More importantly, our data indicated that the melanin production of MC could be suppressed by Teff subsets-related cytokines IFN-γ, IL-17A and IL-22thought down-regulating mRNA expression of MITF and tyrosinase genes in MC. In addition, we also observed that IFN-γ could induce MC apoptosis. Part Ⅰ The Epidemiology of Associated AID and ALD in VV PatientsObjectives:To explore the prevalence of associated other AID and ALD in VV patients.Methods:We collected eligible data from127VV patients by a uniform questionnaire. All the data were statistically analyzed by statistic package of social science (SPSS, version15.0).Results:Out of the127VV patients,20(15.75%) cases reported a history of AID, and27(21.26%) cases reported ALD. The significantly elevated complications among this cohort of VV were autoimmune thyroiditis (13.39%), chronic urticaria (11.02%), asthma (6.30%) and SLE (0.79%), compared with the prevalence of those diseases in the general population (P<0.05).Conclusion:In VV patients, the prevalence of associated additional AID and ALD are significantly higher than those in the general population, which indicates that a systemic immune activation may be involved in the pathogenesis of VV and its associated diseases. Part Ⅱ Teff Subsets and Treg Population Dynamics in VVObjective:To investigate the relative abundances and activities of Teff subsets and Treg in VV.Method:Fifty patients with VV and20healthy individuals were included for blood sample collection, which were processed to detect the proportion of Treg and Teff subsets including T helper (Th)1、Th2、Th17、Th22、T cytotoxic (Tc)1、Tc2、 Tc17、Tc22and classic cytotoxic T lymphocytes (CTL) by flow cytometry. Related cytokines concentration in sera, including IFN-γ、TNF-α、IL-4、IL-17A、IL-22、IL-6、 TGF-β and IL-10were detected by ELISA. Skin biopsies were collected and stained for infiltrating cells with positive expression of CD8、IFN-γ、IL-17A、IL-22or Foxp3by immunohistochemical assessment. The inhibiting function of CD4+CD25+Treg on the proliferation of autologous CD8+T cells or CD4+CD25-Tresp cells were detected by the in vitro co-culture at different ratios.Results:In patients with VV, the percentage of all Tc/CTL subsets, Th17and Th22were significantly higher, while the proportion of Treg was lower compared with those in healthy individuals. The serum levels of IFN-γ, IL-6, IL-17A and IL-22in progressive VV patients were increased, yet that of TGF-β decreased. An immunohistochemical analysis showed increased Teff subsets expressing CD8, IFN-γ, IL-17A or IL-22, and elevated Treg expressing Foxp3were infiltrating in the progressive perilesional skin samples of VV patients. The peripheral Treg of progressive VV patients were impaired in their ability to suppress the proliferation and cytolytic capacity of autologous CD8+T cells and CD4+CD25-Tresp cells.Conclusion:A quantitive and functional failure of Treg may contribute to the hyper-activation of Teff subsets, which may involved in the pathogenesis of W and its associated AID and ALD. Part Ⅲ Induced Dysfunction of MC by Teff Subsets-Related CytokinesObjective:To investigate whether the proinflammatory cytokines produced by Teff subsets influence the viability, enzymatic activity, melanin production and gene expression in MC.Method:MC were treated with the supernatant of stimulated peripheral blood monouclear cells (PBMC), or the supernatant of stimulated CD4+T cells or CD8+T cells, or the supernatant of stimulated CD4+T cells or CD8+T cells with anti-IFN-y antibodies (Abs), anti-interleukin (IL)-17Abs or anti-IL-22Abs, or recombinant human cytokines IFN-γ, IL-17A or IL-22for48hours, respectively. Cell viability was examined by the cell counting kit-8(CCK-8) assay. Cellular tyrosinase activity and melanin content were determined using spectrophotographic methods. Related gene expression was analyzed by real-time reverse transcription-polymerase (PCR).Results:An in vitro analysis showed that the viability, enzymatic activity and melanin production in MC were all suppressed by treatment with the supernatant of stimulated PBMCs and Teff subsets, as well as by IFN-γ, IL-17A and IL-22. The expression of MITF and tyrosinase mRN A were downregulated in MC after treating with IFN-γ, IL-17A and IL-22.Conclusion:Our results provide evidence of the inhibition of Teff subsets on the function of MC in VV by producing increased levels of cytokines IFN-γ, IL-17A and IL-22, which may contribute to the skin hypopigmentation in VV patients. Part Ⅳ Induced Apoptosis of MC by IFN-y, IL-17and IL-22γObjective:To investigate whether the proinflammatory cytokines IFN-y, IL-17A and IL-22induce MC apoptosis.Method:MC were treated with recombinant human cytokines IFN-γ, IL-17A or IL-22for48hours, respectively. Cell morphology were observed by an inverted microscope, nuclear changes were examined by Hoechst33258staining, and the percentage of cell apoptosis were detected by Annexin V/PI double staining flow cytometry.Results:We observed that a high proportion of MC was apoptosis by treatment with IFN-γ, which were identified on cell shrinkage, dendritic disappearance and nuclear fragmentation. Yet no significant difference was detected in the percentage of apoptotic MC between groups treating with IL-17A or IL-22, and those treating with medium.Conclusion:IFN-γ could induce MC apoptosis, which might be involved in the loss of MC and the appearance of white macules in the skin of VV patients.