Mapping architectural and transcriptional alterations in non-lesional and lesional epidermis in vitiligo

Abstract

In vitiligo, chronic loss of melanocytes and consequent absence of melanin from the epidermis presents a challenge for long-term tissue maintenance. The stable vitiligo patches are known to attain an irreversible depigmented state. However, the molecular and cellular processes resulting in this remodeled tissue homeostasis is unclear. To investigate the complex interplay of inductive signals and cell intrinsic factors that support the new acquired state, we compared the matched lesional and non-lesional epidermis obtained from stable non-segmental vitiligo subjects. Hierarchical clustering of genome-wide expression of transcripts surprisingly segregated lesional and non-lesional samples in two distinct clades, despite the apparent heterogeneity in the lesions of different vitiligo subjects. Pathway enrichment showed the expected downregulation of melanogenic pathway and a significant downregulation of cornification and keratinocyte differentiation processes. These perturbations could indeed be recapitulated in the lesional epidermal tissue, including blunting of rete-ridges, thickening of stratum corneum and increase in the size of corneocytes. In addition, we identify marked increase in the putrescine levels due to the elevated expression of spermine/spermidine acetyl transferase. Our study provides insights into the intrinsic self-renewing ability of damaged lesional tissue to restore epidermal functionality in vitiligo.

Figure 1

Dominant signature of keratinocyte pathology in the lesional vitiligo skin. (a) Clustering of samples based on average normalized expression values from microarray segregates NL and L skin (n = 15). Enrichment analysis of top 1% of (b) down regulated and (c) up regulated genes was performed by DAVID bioinformatics resource. Negative log transformed p-value of the enrichment of the pathways was calculated and represented as a bar graph. (d) Transcriptional regulation of genes involved in maintaining epidermal integrity and cell-cell adhesion in keratinocytes. Color scheme-Red- upregulated; green- downregulated; grey- not regulated in lesional as compared to matched non-lesional skin.

Figure 2

Architectural alterations in stable vitiligo lesions. (a) Immunohistochemical staining of melanocyte-specific S100 antigen, Hematoxylin and Eosin staining (H&E), and transmission electron micrographs (TEM) in paired non-lesional (NL) and lesional (L) skin sections. Arrows indicate melanosomes. Scale bar in S100 stained sections is 10 μm, 50 μm for H&E image and 1μm for TEM image. (b) Bar plots depicting architectural features quantitated in skin sections: thickness of stratified epithelia (from stratum basale to stratum corneum), cellular epidermis (from stratum basale to stratum granulosum) and thickness of stratum corneum in μm, ratio of length of secondary (μm) to primary ridge (μm) and the total number of cells per square mm in non-leisonal (NL) and lesional (L) samples (n = 15). The box plot represents the mean ± range of the data. Indicated p-values computed using a paired t-test involving n = 15 (NL vs L) pairs. (c) TEM images of stratum corneum (corneocytes) from non-lesional and lesional epidermis. Magnification is 1700X, scale bar is 0.5μm (d) Quantitation of thickness among three layers of corneocytes close to the viable epidermis across four pairs of matched NL and L skin sections, significance calculated using paired t test.

Figure 3

Altered polyamine metabolism in vitiligo epidermis. (a) Thin layer chromatographic analysis of polyamines in non-lesional and lesional epidermis after dansylation. Whole epidermis was extracted using perchloric acid and the extracts were dansylated along with standard polyamines. The reactions were extracted with toluene and spotted onto silica TLCs and developed on a cyclohexane: ethylacetate solvent system and detected under UV transilluminiscence. The amount of each of the polyamine per milligram of the epidermis is quantitated and represented across three patient samples (3 non-lesional and 4 lesional epidermis). (b) Log transformed relative levels of putrescine (PUT) and spermine (SPN) in three patient samples (3 non-lesional and 4 lesional epidermis) as detected by thin layer chromatography. Dotted line represents unchanged levels of polyamines with respect to the non-lesional skin (c) Real-time PCR analysis of Spermine/spermidine acetyl transferase-1 (SSAT-1) mRNA across five independent pairs of vitiligo lesional and non-lesional skin, represented as a scatter plot, horizontal line indicates mean. PUT- putriscine, SPD- spermidine, SPN- spermine, DAH- 1,7-diaminoheptane. NL- non-lesional, L- lesional skin.

Figure 4

Altered functional networks in vitiligo. Network map of clusters of genes associated with vitiligo predisposition along with their interacting partners were mapped to the differential expression of genes observed in vitiligo. Three networks are shown: SCF-KIT signaling, oxidative stress and immune response. Each node (gene) is colored according to upregulation (red), downregulation (green), or no regulation (grey). White denotes proteins for which corresponding probes were not found in the microarray. The regulation score was calculated from microarray experiments performed on 15 vitiligo samples, where only agreement between >= 11 samples was included as a significant regulation.