BackgroundInterferon-γ (IFN-γ) plays an important role in the proceedings of vitiligo through recruiting lymphocytes to the lesional skin. However, the potential effects of IFN-γ on skin melanocytes and the subsequent contribution to the vitiligo pathogenesis are still unclear.ObjectiveTo investigate the effects of IFN-γ on viability and cellular functions of melanocytes.MethodsPrimary human melanocytes were treated with IFN-γ. Cell viability, apoptosis, cell cycle melanin content and intracellular reactive oxygen species (ROS) level were measured. mRNA expression was examined by real-time PCR. The release of interleukin 6 (IL-6) and heat shock protein 70 (HSP-70) was monitored by ELISA. β-galactosidase staining was utilized to evaluate melanocyte senescence.ResultsPersistent IFN-γ treatment induced viability loss, apoptosis, cell cycle arrest and senescence in melanocytes. Melanocyte senescence was characterized as the changes in pigmentation and morphology, as well as the increase of β-galactosidase activity. Increase of p21Cip1/Waf1 protein was evident in melanocytes after IFN-γ treatment. IFN-γ induction of senescence was attenuated by siRNAs against p21, Janus kinase 2 (JAK2) or signal transducer and activator of transcription 1 (STAT1), but not by JAK1 siRNA nor by p53 inhibitor pifithrin-α. IFN-γ treatment increased the accumulation of intracellular ROS in melanocytes, while ROS scavenger N-acetyl cysteine (NAC) effectively inhibited IFN-γ induced p21 expression and melanocyte senescence. IL-6 and HSP-70 release was significantly induced by IFN-γ treatment, which was largely inhibited by NAC. The increase of IL-6 and HSP-70 release could also be observed in senescent melanocytes.ConclusionIFN-γ can induce senescence in melanocytes and consequently enhance their immuno-competency, leading to a vitiligo-prone milieu.

We here investigated the efficiency of autologous melanocyte transplantation of 23 vitiligo patients by focusing on perilesional skin homing CD8+ T lymphocytes, and studied the potential effect of dermal mesenchymal stem cells (DMSCs) on CD8+ T cell activities in vitro. Out of 23 patients with the autologous melanocyte transplantation, 12 patients (52.17%) had an excellent re-pigmentation, 6 patients (26.09%) had a good re-pigmentation, 5 patients (21.74%) had a fair or poor re-pigmentation. CD8+ T cells infiltrating was observed in the perilesional vitiligo area of all patients. Importantly, the efficiency of the transplantation was closely associated with skin-homing CD8+ T cell activities. The patients with high number of perilesional CD8+ T cells or high level of cytokines/chemokines were associated with poor re-pigmentation efficiency. For in-vitro experiments, we successfully isolated and characterized human DMSCs and skin-homing CD8+ T cells. We established DMSCs and CD8+ T cell co-culture system, where DMSCs possessed significant inhibitory effects against skin homing CD8+ T lymphocytes. DMSCs inhibited CD8+ T cells proliferation, induced them apoptosis and regulated their cytokines/chemokines production. Our results suggest that vitiligo patients’ autologous melanocytes transplantation efficiency might be predicted by perilesional skin-homing CD8+ T cell activities, and DMSCs might be used as auxiliary agent to improve transplantation efficacy.

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The paucity of vitiligo animal models limits the understanding of vitiligo pathogenesis and the development of therapies for the skin disorder. In this study, we developed a new mouse model of vitiligo by topically applying the skindepigmenting agent monobenzone on mice. We demonstrated that monobenzone-induced skin depigmentation on the nonexposed sites and that the severity of lesions depended on drug dosage. The result of the histological examination of the depigmented skin indicated loss of epidermal melanocytes and perilesional accumulation of CD8 + T cells. Furthermore, the monobenzone-induced depigmentation of the Rag1 gene knockout did not appear on the non-exposed sites, supporting the involvement of infiltrating CD8 + T cells in melanocyte destruction. Resemblance in histological characteristics and pathogenesis between monobenzone-induced depigmentation and active human vitiligo suggests good potential of our mouse model for use in vitiligo research.

Considerable evidence implicates that viral infection might be a participant factor in the pathogenesis of vitiligo. However, it is still unclear how viral infection leads to the melanocyte destruction. To elucidate the effects of viral dsDNA on the viability and cytokine synthesis of normal human melanocytes and to explore the underlying mechanisms, primary cultured normal human melanocytes were transfected with poly (dA:dT). The results demonstrated that poly(dA:dT) triggered apoptosis instead of pyroptosis in melanocytes. Knocking down AIM2 or RIG-I by RNA interference partially reduced the poly (dA:dT)-induced LDH release, suggesting the involvement of both nucleic acid sensors in the process of melanocyte death. Poly(dA: dT) induced the expression of pro-inflammatory cytokine genes including IFN-b, TNF-a, IL-6 and IL-8 as well, whereas the pro-inflammatory cytokine production was suppressed by RIG-I siRNA, but not by AIM2 siRNA. Poly(dA:dT) treatment increased the phosphorylation of p38 and JNK and NFjB. Accordingly, NFjB inhibitor Bay 11-7082 and JNK inhibitor SP600125 blocked the induction of the cytokine genes except IFN-b. The production of IL6 and IL8 was also suppressed by p38 inhibitor SB203580. On the contrary, the Poly(dA:dT)-induced melanocyte death was only decreased by SP600125. This study provides the possible mechanism of melanocyte destruction and immuno-stimulation in vitiligo by innate immune response following viral infection.

Vitiligo is an inflammatory skin disorder in which activated T cells play an important role in its onset and progression. Epigallocatechin-3-gallate (EGCG), the major chemical constituent of green tea, exhibits remarkable anti-oxidative and anti-inflammatory properties. EGCG administration has been confirmed to decrease the risk of vitiligo; however, the underlying mechanism is undetermined. In this study, we proved that EGCG directly inhibited the kinase activity of Janus kinase 2 (JAK2). In primary cultured human melanocytes, EGCG pre-treatment attenuated interferon ( Key words vitiligo; epigallocatechin-3-gallate; melanocyte; Janus kinase 2; chemoattractant Vitiligo is a depigmentation disorder caused by the destruction of epidermal melanocytes.1) The exact pathophysiological mechanism of vitiligo remains elusive. However, several hypotheses, including autoimmune, oxidant-antioxidant, genetic susceptibility, neural and viral mechanisms, have been proposed to explain the selective destruction of melanocytes. Currently, clinical and bench findings suggest the major role of autoimmune factors in the progress of vitiligo. Inflammatory cells, mostly T lymphocytes, have been identified close to vitiligous skin lesion.2,3) Lesional CD8+ T cells specially induce melanocyte apoptosis in unaffected skin ex vivo, and the frequency of anti-melanocyte CD8+ T cells in both the blood and skin of vitiligo patients correlates with the severity of disease. 4,5) Moreover, immunosuppressive therapies targeting T-cell activation have been shown to be effective for vitiligo treatment.6,7) These evidences all support a direct role for T lymphocytes in melanocyte destruction in human vitiligo.Epigallocatechin-3-gallate (EGCG), a major catechin in green tea, is considered beneficial for human health, especially as an anti-oxidative agent.8) In addition, EGCG was confirmed to directly inhibit protein kinases by working as an ATP analog.9) Previously, we reported the therapeutic effect of EGCG in vitiligo induced by monobenzone in mice. 10) Our results suggested that it could contribute to the suppression of CD8+ T cell migration and the inflammatory cytokine expression. However, the mechanism regarding the EGCG regulation of immune responses in vitiligo is still unclear.The aim of study is to elucidate the inhibitory effects of EGCG on inflammatory signaling pathways and to identify the target of EGCG inhibition. RESULTS AND DISCUSSION EGCG Inhibited Janus Kinase (JAK)2 Activity in VitroThe JAKs are a family of four non-receptor tyrosine kinases including JAK1, JAK2 JAK3 and tyrosine kinase (TYK)2. Recruitment of stimuli to cell surface receptors activates JAKs which, in turn, phosphorylates and stimulates latent cytoplasmic signal transducer and activator of transcription (STAT) proteins to an active dimer, leading to nuclear translocation and DNA binding and subsequently modulating gene transcription.11) The JAK/STAT signal pathway controls a number of important biological responses, including immune functions, cellular growth, cell...

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Background:Vitiligo is an acquired pigmentary disorder. In vivo reflectance confocal microscopy (RCM) reproducible imaging technique has already been reported to be useful in the diagnosis of other skin diseases.Objective:To define RCM features of vitiligo on different clinical stages.Materials and Methods:A total of 125 patients with a clinical diagnosis of vitiligo were included in this study. After informed consent, lesional skins of those vitiligo patients were characterized by using RCM. Five patients with inflammatory cell infiltration observed at the edge of skin lesions and another 5 patients without inflammatory cell infiltration were selected. Biopsies were performed at same sites of the RCM examination areas for histological and immune-histological analysis.Results:In the active stage of vitiligo, the RCM examination revealed that the bright dermal papillary rings presented at the dermoepidermal junction level in normal skin lost their integrity or totally disappeared, border between vitiligo lesion and normal skin became unclear, and highly refractile cells that referred to infiltrated inflammatory cells could be seen within the papillary dermis at the edge of the lesions. In the stable stage of vitiligo, the RCM showed a complete loss of melanin in lesional skin and a clear border between lesional and normal skin.Conclusion:A simple clinical examination with RCM may reliably and efficiently allow evaluation of the stability status of vitiligo lesions.