Transcriptome analysis reveals markers of aberrantly activated innate immunity in vitiligo lesional and non-lesional skin

Richard Yu, Raewyn Broady, Yuanshen Huang, Yang Wang, Jie Yu, Min Gao, Megan Levings, Shencai Wei, Shengquan Zhang, Aie Xu, Mingwan Su, Jan Dutz, Xuejun Zhang, Youwen Zhou, Richard Yu, Raewyn Broady, Yuanshen Huang, Yang Wang, Jie Yu, Min Gao, Megan Levings, Shencai Wei, Shengquan Zhang, Aie Xu, Mingwan Su, Jan Dutz, Xuejun Zhang, Youwen Zhou

Abstract

Background: Vitiligo is characterized by the death of melanocytes in the skin. This is associated with the presence of T cell infiltrates in the lesional borders. However, at present, there is no detailed and systematic characterization on whether additional cellular or molecular changes are present inside vitiligo lesions. Further, it is unknown if the normal appearing non-lesional skin of vitiligo patients is in fact normal. The purpose of this study is to systematically characterize the molecular and cellular characteristics of the lesional and non-lesional skin of vitiligo patients.

Methods and materials: Paired lesional and non-lesional skin biopsies from twenty-three vitiligo patients and normal skin biopsies from sixteen healthy volunteers were obtained with informed consent. The following aspects were analyzed: (1) transcriptome changes present in vitiligo skin using DNA microarrays and qRT-PCR; (2) abnormal cellular infiltrates in vitiligo skin explant cultures using flow cytometry; and (3) distribution of the abnormal cellular infiltrates in vitiligo skin using immunofluorescence microscopy.

Results: Compared with normal skin, vitiligo lesional skin contained 17 genes (mostly melanocyte-specific genes) whose expression was decreased or absent. In contrast, the relative expression of 13 genes was up-regulated. The up-regulated genes point to aberrant activity of the innate immune system, especially natural killer cells in vitiligo. Strikingly, the markers of heightened innate immune responses were also found to be up-regulated in the non-lesional skin of vitiligo patients.

Conclusions and clinical implications: As the first systematic transcriptome characterization of the skin in vitiligo patients, this study revealed previously unknown molecular markers that strongly suggest aberrant innate immune activation in the microenvironment of vitiligo skin. Since these changes involve both lesional and non-lesional skin, our results suggest that therapies targeting the entire skin surface may improve treatment outcomes. Finally, this study revealed novel mediators that may facilitate future development of vitiligo therapies.

Conflict of interest statement

Competing Interests: This work was in part supported by funding from Astellas Pharma Research Competition, Canada. However, this does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Transcriptome Analysis of Vitiligo and…
Figure 1. Transcriptome Analysis of Vitiligo and Normal Skin Biopsies.
A heat map is constructed by Gene Spring software (see methods) comparing the relative expression levels of the 30 significantly altered genes in vitiligo skin. Depicted are the expression levels of these genes in individual samples relative to their corresponding expression reference levels, which are the averages of expression in the 16 normal skin biopsies. Red squares: Genes with up-regulation in that sample compared with normal skin of healthy volunteers. Green squares: Genes with down-regulation in that specific sample compared with normal skin of healthy volunteers. Yellow squares: no significant change between the sample and the normal skin of healthy volunteers.
Figure 2. Explant culture analysis of natural…
Figure 2. Explant culture analysis of natural killer cell infiltrates in biopsies of vitiligo lesional and non-lesional skin.
Natural killer (NK) cells from 6 pairs of vitiligo skin explants (lesional and non-lesional) and 5 normal skin explants were cultured on Cellfoam matrices (see methods section) and analyzed using flow cytometry, with the gate set on total live cells A: Skin-resident CD56bright CD3-ve NK cells in normal control skin, vitiligo non-lesional skin and lesional skin by scatter plot. B: Further gating on the CD56bright CD3-ve cells revealed that majority of the NK cells in vitiligo skin were granzyme B-positive. C: Dot plot of all samples analyzed for CD56bright CD3- natural killer cells. The difference in the proportion of resident natural killer cells between normal skin and the respective vitiligo non-lesional and lesional skin is statistically significant (p = 0.0043; mean ± SEM). Comparisons between the respective groups are indicated in the figure by lines with an asterisk (*) denoting statistical significance (p<0.05). Abbreviations: NS: normal skin; NLS: non-lesional skin; LS: lesional-skin.
Figure 3. Distribution of natural killer cells…
Figure 3. Distribution of natural killer cells and cytotoxic T cells in vitiligo lesional and non-lesional skin.
Skin biopsies taken from 12 vitiligo patients and 6 normal individuals were subjected to immunofluorescence analysis of natural killer (NK) cells. A: Micrographs showing natural killer cells (CD3−/NKG2D+) (red) present in vitiligo lesional and non-lesional skin but absent from the normal skin of healthy volunteers. Some NK cells are in close proximity to the basal epidermal layer where melanocytes reside (arrows). In addition, increased numbers of cytotoxic T cells (CD3+/NKG2D+) (yellow: co-localization of red and green) as well as non-cytotoxic T cells (CD3+/NKG2D−) (green) were also found in both vitiligo peri-lesional and lesional skin. B: Quantification of cells demonstrates a statistically significant increase in NK cells, cytotoxic T cells and non-cytotoxic T cells in vitiligo non-lesional skin (p = 0.0021, 0.0015, 0.001; mean ± SEM) and lesional skin (p = 0.021, 0.0017, 0.0023; mean ± SEM) as compared with normal skin. Color keys: Green: CD3 (a pan-T cell marker); Red: NKG2D (NK cell activation receptor); and blue: DAPI (nuclear stain). Comparisons between the respective groups are indicated in the figure by lines with an asterisk (*) denoting statistical significance (p<0.05). Abbreviations: NS: normal skin; NLS: non-lesional skin; LS: lesional-skin. Magnification: 400×; scale bar: 20 µm.

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