Vitiligo Epidermis Research Articles

Serotonin/5-HT7 receptor provides an adaptive signal to enhance pigmentation response to environmental stressors through cAMP-PKA-MAPK, Rab27a/RhoA, and PI3K/AKT signaling pathways.

Serotonin (5-HT), a neurotransmitter, is essential for normal and pathological pigmentation processing, and its receptors may be therapeutical targets. The effect and behavior of the 5-HT7 receptor (5-HT7R) in melanogenesis in high vertebrates remain unknown. Herein, we examine the role and molecular mechanism of 5-HT7R in the pigmentation of human skin cells, human tissue, mice, and zebrafish models. Firstly, 5-HT7R protein expression decreased significantly in stress-induced depigmentation skin and vitiligo epidermis. Stressed mice received transdermal serotonin 5-HT7R selective agonists (LP-12, 0.01%) for 12 or 60 days. Mice might recover from persistent stress-induced depigmentation. The downregulation of tyrosinase (Tyr), microphthalmia-associated transcription factor (Mitf) expression, and 5-HT7R was consistently restored in stressed skin. High-throughput RNA sequencing showed that structural organization (dendrite growth and migration) and associated pathways were activated in the dorsal skin of LP-12-treated animals. 5-HT7R selective agonist, LP-12, had been demonstrated to enhance melanin production, dendrite growth, and chemotactic motility in B16F10 cells, normal human melanocytes (NHMCs), and zebrafish. Mechanistically, the melanogenic, dendritic, and migratory functions of 5-HT7R were dependent on the downstream signaling of cAMP-PKA-ERK1/2, JNK MAPK, RhoA/Rab27a, and PI3K/AKT pathway activation. Importantly, pharmacological inhibition and genetic siRNA of 5-HT7R by antagonist SB269970 partially/completely abolished these functional properties and the related activated pathways in both NHMCs and B16F10 cells. Consistently, htr7a/7b genetic knockdown in zebrafish could blockade melanogenic effects and abrogate 5-HT-induced melanin accumulation. Collectively, we have first identified that 5-HT7R regulates melanogenesis, which may be a targeted therapy for pigmentation disorders, especially those worsened by stress.

Integrated analysis of miRNA-mRNA networks reveals a strong anti-skin cancer signature in vitiligo epidermis.

Expression of microRNAs (miRNAs) is often dysregulated in several cancers, including non-melanoma skin cancer (NMSC). Individuals with vitiligo possess a deregulated miRnome along with a lower risk of developing NMSCs. We used data sets from our previously published studies on vitiligo epidermis to construct functional miRNA-mRNA networks to understand the molecular basis underlying the lower incidence of NMSC observed in individuals with vitiligo. miRTarBase database was used to fetch the experimentally validated targets of differentially expressed miRNAs and two protein-protein interaction (PPI) networks were constructed for the miRNA-mRNA interactions (230 downregulated targets of 5 upregulated miRNAs and 47 upregulated mRNAs targeted by 12 downregulated miRNAs). Pathway enrichment analysis identified RNA biogenesis and transport as well as cell adhesion to be perturbed in vitiligo. Further, oncogenic transcription factors (OTFs) that were upregulated in publicly available squamous cell carcinoma (SCC) or basal cell carcinoma (BCC) microarray data were compared with that of vitiligo to decode skin cancer-specific molecular signatures. We identified three significantly upregulated miRNAs, miR-31-5p, miR-31-3p and miR-194-3p in lesional epidermis that could negatively regulate seven oncogenic transcription factors, FOXC1, AR, SP1, YY1, GLI2, TP53 and RARA, known to be over-expressed in SCC or BCC. Taken together, our study identified a perturbed miRNA-regulated transcriptome, which potentially confers protection to vitiligo skin from an increased incidence of NMSC.

The long noncoding RNA MALAT1 suppresses miR-211 to confer protection from ultraviolet-mediated DNA damage in vitiligo epidermis by upregulating sirtuin 1.

The absence of melanocytes poses a challenge for long-term tissue homeostasis in vitiligo. Surprisingly, while individuals with Fitzpatrick phototypes I-II (low melanin content) have a higher incidence of melanoma and nonmelanoma skin cancer, people with vitiligo are at a decreased risk for the same. To understand the molecular mechanisms that protect vitiligo skin from ultraviolet (UV)-induced DNA damage by (i) characterizing differentially expressed microRNAs in lesional vs. nonlesional epidermis and (ii) identifying their upstream regulators and downstream gene targets. Genome-wide microRNA profiling of nonlesional and lesional epidermis was performed on five individuals with stable nonsegmental vitiligo using next-generation RNA sequencing. The relevance of the upstream regulator and downstream target gene of the most differentially expressed microRNA was studied. Our study found sirtuin1 (SIRT1), an NAD-dependent deacetylase, to be a direct target of miR-211 - the most significantly downregulated microRNA in lesional epidermis. Inhibition of SIRT1 with EX-527 downregulated keratin 10 and involucrin, suggesting that SIRT1 promotes keratinocyte differentiation. Overexpression of miR-211 mimic led to a significant increase in γ-H2AX positivity and cyclobutane pyrimidine dimer (CPD) formation, hallmarks of UVB-mediated DNA damage. These effects could be ameliorated by the addition of resveratrol, a SIRT1 activator. Furthermore, a long noncoding RNA, MALAT1, was identified as a negative upstream regulator of miR-211. Overexpression of MALAT1 resulted in increased expression of SIRT1 and a concomitant removal of UVB-induced CPDs in primary keratinocytes. These findings establish a novel MALAT1-miR-211-SIRT1 signalling axis that potentially confers protection to the 'amelanotic' keratinocytes in vitiligo.

Oxidative stress-induced IL-15 trans-presentation in keratinocytes contributes to CD8+ T cells activation via JAK-STAT pathway in vitiligo

Oxidative stress and effector memory CD8+ T cells have been greatly implicated in vitiligo pathogenesis. However, the crosstalk between these two crucial pathogenic factors has been merely investigated. IL-15 has been regarded as an important cytokine exerting its facilitative effect on memory CD8+ T cells function in various autoimmune diseases. In the present study, we initially discovered that the IL-15 expression was significantly increased in vitiligo epidermis and highly associated with epidermal H2O2 content. In addition, epidermal IL-15 expression was mainly derived from keratinocytes. Then, we showed that oxidative stress promoted IL-15 and IL-15Rα expression as well as IL-15 trans-presentation by activating NF-κB signaling in keratinocytes. What's more, the trans-presented IL-15, rather than the secreted one, was accounted for the potentiation of CD8+ TEMs activation. We further investigated the mechanism underlying trans-presented IL-15 in potentiating CD8+ TEMs activation and found that the blockage of IL-15-JAK-STAT signaling could be a potent therapeutic approach. Taken together, our results demonstrate that oxidative stress-induced IL-15 trans-presentation in keratinocytes contributes to the activation of CD8+ TEMs, providing a novel mechanism by which oxidative stress initiates autoimmunity in vitiligo.

792 Oxidative stress-induced IL-15 trans-presentation in keratinocytes contributes to CD8+ T cells activation via JAK-STAT pathway in vitiligo

Oxidative stress and melanocyte-specific CD8+ T cell have been greatly implicated in vitiligo pathogenesis. However, the crosstalk between these two crucial pathogenic factors has been merely investigated. IL-15 has been regarded as an important cytokine exerting its facilitative effect on immune cell function in various autoimmune diseases. In the present study, we initially discovered that the IL-15 expression was significantly increased in vitiligo epidermis and highly associated with epidermal H2O2 content. In addition, epidermal IL-15 expression was mainly derived from keratinocytes. Then, we showed that oxidative stress promoted IL-15 and IL-15Rα expression as well as IL-15 trans-presentation by activating NF-κB signaling in keratinocyte. What’s more, the trans-presented IL-15, rather than the secreted one, was accounted for the potentiation of CD8+ T cells activation. We further investigated the mechanism underlying trans-presented IL-15 in potentiating CD8+ T cell activation and found that the blockage of IL-15-JAK-STAT signaling could be a potent therapeutic approach. Taken together, our results demonstrate that oxidative stress-induced IL-15 trans-presentation in keratinocyte contributes to the activation of CD8+ T cell, providing a novel mechanism by which oxidative stress initiates autoimmunity in vitiligo.

Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from melanocyte and adjacent keratinocyte populations, of satisfactory quality RNA that was successfully amplified and analysed by qRT-PCR. The melanocyte-specific gene transcripts TYR, DCT, TYRP1 and PMEL were significantly upregulated in our NBUVB-treated melanocyte samples as compared with the keratinocyte samples, while keratinocyte-specific genes (KRT5 and KRT14) were expressed significantly higher in the keratinocyte samples as compared with the melanocyte samples. Furthermore, in both NBUVB-treated vitiligo skin and normal skin, when bulge melanocytes were compared with epidermal melanocytes, we found significantly lower expression of melanocyte-specific genes and significantly higher expression of three melanocytic stem cell genes (SOX9, WIF1 and SFRP1), while ALCAM and ALDH1A1 transcripts did not show significant variation. We found significantly higher expression of melanocyte-specific genes in the epidermis of NBUVB-treated vitiligo, as compared to the normal skin. When comparing bulge melanocyte samples from untreated vitiligo, NBUVB-treated vitiligo and normal skin, we did not find significant differences in the expression of melanocyte-specific genes or melanocytic stem cell genes. These techniques offer valuable opportunities to study melanocytes and their precursors in vitiligo and other pigmentation disorders.

Immunohistochemical expression of AQP-3 in vitiligo: a new potential guide for disease activity.

Vitiligo is a depigmenting skin disorder, with disappearance of functioning epidermal melanocytes. Aquaporin-3 (AQP-3) is an aquaglyceroporin expressed in epidermal keratinocytes, where it shares in regulating their proliferation and differentiation, and so it might affect melanocytes indirectly. So far, little is known regarding its possible role in vitiligo. This work aimed to study the changes in immunohistochemical expression of AQP-3 protein in vitiligo to detect its possible role in disease pathogenesis. Skin biopsies were taken from lesional skin of 30 vitiligo patients in addition to 20 normal controls. Epidermal immunohistochemical expression of AQP-3 was assessed as: +3 = strong expression, +2 = moderate, +1= weak and 0= negative expression. AQP-3 was significantly less expressed in vitiligo epidermis than control (P<0.001) with an inverse correlation with Vitiligo Index of Disease Activity (r =-0.505, P=0.004). Reduced epidermal AQP-3 may have a role in impaired melanocyte survival in vitiligo, and might be a potential negative biological marker for vitiligo activity. Larger trials should further elucidate the effect of changes in epidermal AQP-3 expression in development of vitiligo, and that might pave the road for discovering new therapeutic modalities for the disease.

Vitiligo-Inducing Phenols Activate the Unfolded Protein Response in Melanocytes Resulting in Upregulation of IL6 and IL8

Vitiligo is characterized by depigmented skin patches due to loss of epidermal melanocytes. Oxidative stress may play a role in vitiligo onset, while autoimmunity contributes to disease progression. In this study we sought to identify mechanisms that link disease triggers and spreading of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress results in redox disruptions that extend to the ER, causing accumulation of misfolded peptides, which activates the unfolded protein response (UPR). We used 4-tertiary butyl phenol (4-TBP) and monobenzyl ether of hydroquinone (MBEH), known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box binding protein 1 (XBP1), are increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators interleukin-6 (IL6) and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while over-expression of XBP1 alone increased their expression. Thus, melanocytes themselves produce cytokines associated with activation of an immune response following exposure to chemical triggers of vitiligo. These results expand our understanding of the mechanisms underlying melanocyte loss in vitiligo and pathways linking environmental stressors and autoimmunity.

Repigmentation after epidermal grafting of vitiligo epidermis into normal skin in segmental vitiligo

Repigmentation after epidermal grafting of vitiligo epidermis into normal skin in segmental vitiligo

Keratinocyte dysfunction in vitiligo epidermis: cytokine microenvironment and correlation to keratinocyte apoptosis.

Vitiligo is a skin disorder characterized by loss of functional melanocytes. Keratinocytes contribute to melanocyte homeostasis, and keratinocyte alteration may play a role in melanocyte dysfunction in vitiligo. In particular, the release of melanogenic mediators and the level of functioning keratinocytes may affect melanocyte dysfunction in vitiligo epidermis. Keratinocyte-derived mediators involved in pigmentation, analysed by in situ hybridization, and epidermal apoptosis, detected by TUNEL assay and electron microscopy, were evaluated in lesional and perilesional skin biopsies from 15 patients with active vitiligo and in 5 control subjects. Among the melanogenic mediators, stem cell factor (SCF) and endothelin-1 (ET-1) mRNA were significantly reduced in lesional as compared to perilesional epidermis, whereas no difference was observed in mRNA of basic fibroblastic growth factor (bFGF) and granulocyte-monocyte colony stimulating factor (GM-CSF). The expression of mRNA for tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6), two pro-inflammatory cytokines with an inhibitory effect on pigmentation, was increased in the epidermis from vitiligo biopsies, whereas their expression was practically undetectable in the skin of control subjects. Apoptotic keratinocytes were more abundant in lesional vs. perilesional skin of vitiligo patients and were absent in the epidermis of control subjects. Changes in expression of keratinocyte-derived mediators observed in the present study are consistent with their differential functions in melanocyte regulation. In particular, increased TNF-alpha could contribute to keratinocyte apoptosis, which results in reduced release of melanogenic cytokines and ultimately in melanocyte disappearance.

Mechanisms underlying the dysfunction of melanocytes in vitiligo epidermis: role of SCF/KIT protein interactions and the downstream effector, MITF-M.

Little is known about the mechanisms involved in the dysfunction of melanocytes in vitiligo epidermis. It is hypothesized that some cytokine/receptor interactions may be affected, resulting in dysfunction and/or loss of melanocytes. This study has compared the expression of endothelin (ET)-1, the ET-1 receptor (ET(B)R), granulocyte macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), the SCF receptor (KIT protein), tyrosinase, and S100 alpha between lesional and non-lesional vitiligo epidermis. Analysis by reverse transcription-polymerase chain reaction (RT-PCR) and by western blotting for ET-1 and SCF unexpectedly demonstrated up-regulated expression of these cytokines in lesional vitiligo epidermis. Immunohistochemistry with antibodies to melanocyte markers revealed that at the edge of the lesional epidermis, melanocytes remain and express tyrosinase, S100 alpha and ET(B)R, but not KIT protein or melanocyte-specific microphthalmia-associated transcription factor (MITF-M). Quantitation of the staining revealed a slight or moderate decrease in the number of S100 alpha, tyrosinase, and ET(B)R-positive cells at the edge of the lesional epidermis. In contrast, the number of cells expressing KIT protein was markedly decreased at the edge of the lesional epidermis compared with the non-lesional epidermis. At the centre of the lesional epidermis, there was complete loss of melanocytes expressing KIT protein, S100 alpha, ET(B)R, and/or tyrosinase. Western blotting revealed down-regulated expression of c-kit and MITF-M proteins at the edge of the lesional epidermis in vitiligo. These findings suggest that reduction in the expression of KIT protein by melanocytes and its downstream effectors, including MITF-M, may be associated with the dysfunction and/or loss of melanocytes in vitiligo epidermis.

Epidermal oxidative stress in vitiligo.

Epidermal levels of enzymatic and non-enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), vitamin E (Vit E), ubiquinol (CoQ10H2), and reduced glutathione (GSH), as well as polyunsaturated fatty acids of phospholipids (PL-PUFA), were evaluated in the affected epidermis of 15 patients with active vitiligo (AVP) and in the corresponding epidermis of 15 healthy phototype matched controls. The epidermal levels of CoQ10H2, Vit E, GSH, and CAT activity were significantly reduced in AVP and were associated with a marked increase of oxidized glutathione, whereas SODs and GSH-Px activities and ubiquinone concentration remained similar to control values. Antioxidant deficiency, in particular the decline of lipophilic antioxidants, i.e., CoQ10H2 and Vit E, accounts well for PL-PUFA reduction observed in vitiligo epidermis, mainly affecting C18:3 n-3, C20:3 n-6, C20:4 n-6, and C22:6 n-3 fatty acids and suggesting the occurrence of a lipoperoxidative process. In conclusion, both an imbalance of the intracellular redox status and a significant depletion of enzymatic and non-enzymatic antioxidants feature the epidermis of AVP, and represent a fingerprint of an abnormal oxidative stress leading to epidermal cell injury.

Relative Contributions of Decay Accelerating Factor (DAF), Membrane Cofactor Protein (MCP) and CD59 in the Protection of Melanocytes from Homologous Complement

Complement regulatory molecules, membrane cofactor protein (MCP), decay accelerating factor (DAF) and CD59, protect body cells from autologous complement. They have wide tissue distribution but nothing is known about the expression of these molecules on human melanocytes. Since melanocytes are lysed in the lesional skin of patients with a depigmentary disorder vitiligo, it is important to compare the protection offered by complement regulatory molecules to melanocytes present in normal and vitiligo epidermis, against autologous complement. From this point of view, we investigated the differential expression of MCP, DAF and CD59 on normal cultured human melanocytes and assessed their individual contribution in the protection of these cells against complement-mediated damage. Flow cytometric analysis showed that MCP and DAF but not CD59 were expressed on cultured melanocytes. When heat inactivated sera of patients with vitiligo were used as a source of anti-melanocyte antibody to sensitize melanocytes, and guinea pig serum (GpS) or normal human serum (NHS) as a source of complement, GpS was found to be more effective in causing the lysis of melanocytes than NHS. When melanocytes were sensitized with autoantibody as well as F(ab') 2 fragment of either anti-MCP or anti-DAF and subsequently incubated with NHS or GpS, both antibody fragments increased the killing of melanocytes by NHS as well as by GpS. F(ab') 2 fragment of anti-DAF was much more effective in causing enhancement of lysis than that of anti-MCP. Thus, cultured normal human melanocytes express functionally active MCP and DAF but not CD59. Contribution of DAF in protecting melanocytes against complement attack was much more than that of MCP

Epidermal reconstructs in vitiligo: an extrinsic factor is needed to trigger the disease

The primary cellular or molecular targets accounting for melanocytes loss in vitiligo are not clearly identified. To study a putative latent epidermal defect in the epidermis of vitiligo patients, we performed in vitro studies using cultured vitiligo melanocytes and keratinocytes transplanted on to a dead de-epidermized dermis according to a variant of Pruniéras' technique. Control autologous constructs were made with keratinocytes and melanocytes of normal adult epidermis and vitiligo epidermis from perilesional skin. For heterologous reconstructs we combined vitiligo-derived melanocytes or keratinocytes with their normal phototype-matched counterpart. After 15 days of culture at the air-liquid interface, epidermal reconstructs were studied macroscopically and microscopically. Immunohistochemistry was performed using antibodies to TRP-1 and NKI-beteb. All heterologous and autologous reconstructs made with melanocytes and keratinocytes from vitiligo patients had a normal histology and ultrastructure. For vitiligo melanocytes or normal melanocytes submitted to the influence of vitiligo keratinocytes, immunophenotype and function (pigment production and transfer) were similar to normal controls. So, without additional noxious stimuli, we could not discriminate between melanocytes and keratinocytes as inducers of the disease. Our data suggest that the basic abnormality in vitiligo vulgaris needs extrinsic factors to be macroscopically revealed or requires a longer period of culture to develop. Our model will allow analysis of the various pathophysiological mechanisms of vitiligo, e.g. autoantibodies or oxidative stress, at the cellular, biochemical or molecular level.

Melanocyte growth factor is not decreased in vitiligo epidermis

Melanocyte growth factor is not decreased in vitiligo epidermis