Vitiligo non-responding lesions to narrow band UVB have intriguing cellular and molecular abnormalities that may prevent epidermal repigmentation.

We have discovered that human vitiligo patients treated with narrow-band UVB (NBUVB) demonstrated localized resistance to repigmentation in skin sites characterized by distinct cellular and molecular pathways. Using immunostaining studies, discovery-stage RNA-Seq analysis, and confirmatory in situ hybridization, we analyzed paired biopsies collected from vitiligo lesions that did not repigment after 6 months of NBUVB treatment (non-responding) and compared them with repigmented (responding) lesions from the same patient. Non-responding lesions exhibited acanthotic epidermis, had low number of total, proliferative, and differentiated melanocyte (MC) populations, and increased number of senescent keratinocytes (KCs) and of cytotoxic CD8+ T cells as compared with responding lesions. The abnormal response in the non-responding lesions was driven by a dysregulated cAMP pathway and of upstream activator PDE4B, and of WNT/ß-catenin repigmentation pathway. Vitiligo-responding lesions expressed high levels of WNT10B ligand, a molecule that may prevent epidermal senescence induced by NBUVB, and that in cultured melanoblasts prevented the pro-melanogenic effect of α-MSH. Understanding the pathways that govern lack of NBUVB-induced vitiligo repigmentation has a great promise in guiding the development of new therapeutic strategies for vitiligo.

Repigmentation of Human Vitiligo Skin by NBUVB Is Controlled by Transcription of GLI1 and Activation of the ß-Catenin Pathway in the Hair Follicle Bulge Stem Cells.

Vitiligo repigmentation is a complex process in which the melanocyte-depleted interfollicular epidermis is repopulated by melanocyte precursors from hair follicle bulge that proliferate, migrate, and differentiate into mature melanocytes on their way to the epidermis. The strongest stimulus for vitiligo repigmentation is narrow-band UVB (NBUVB), but how the hair follicle melanocyte precursors are activated by UV light has not been extensively studied. To better understand this process, we developed an application that combined laser capture microdissection and subsequent whole transcriptome RNA sequencing of hair follicle bulge melanocyte precursors and compared their gene signatures to that of regenerated mature epidermal melanocytes from NBUVB-treated vitiligo skin. Using this strategy, we found up-regulation of TNC, GJB6, and THBS1 in the hair follicle bulge melanocytes and of TYR in the epidermal melanocytes of the NBUVB-treated vitiligo skin. We validated these results by quantitative real-time-PCR using NBUVB-treated vitiligo skin and untreated normal skin. We also identified that GLI1, a candidate stem cell-associated gene, is significantly up-regulated in the melanocytes captured from NBUVB-treated vitiligo bulge compared with untreated vitiligo bulge. These signals are potential key players in the activation of bulge melanocyte precursors during vitiligo repigmentation.

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.

Narrow Band Ultraviolet B Treatment for Human Vitiligo Is Associated with Proliferation, Migration, and Differentiation of Melanocyte Precursors.

In vitiligo, the autoimmune destruction of epidermal melanocytes produces white spots that can be repigmented by melanocyte precursors from the hair follicles, following stimulation with UV light. We examined by immunofluorescence the distribution of melanocyte markers (C-KIT, DCT, PAX3, and TYR) coupled with markers of proliferation (KI-67) and migration (MCAM) in precursors and mature melanocytes from the hair follicle and the epidermis of untreated and narrow band UVB (NBUVB)-treated human vitiligo skin. NBUVB was associated with a significant increase in the number of melanocytes in the infundibulum and with restoration of the normal melanocyte population in the epidermis, which was lacking in the untreated vitiligo. We identified several precursor populations (melanocyte stem cells, melanoblasts, and other immature phenotypes), and progressively differentiating melanocytes, some with putative migratory and/or proliferative abilities. The primary melanocyte germ was present in the untreated and treated hair follicle bulge, whereas a possible secondary melanocyte germ composed of C-KIT+ melanocytes was found in the infundibulum and interfollicular epidermis of UV-treated vitiligo. This is an exceptional model for studying the mobilization of melanocyte stem cells in human skin. Improved understanding of this process is essential for designing better treatments for vitiligo, ultimately based on melanocyte stem cell activation and mobilization.