Spontaneously Resolved Atopic Dermatitis Shows Melanocyte and Immune Cell Activation Distinct From Healthy Control Skin

Abstract

Atopic dermatitis (AD) typically starts in infancy or early childhood, showing spontaneous remission in a subset of patients, while others develop lifelong disease. Despite an increased understanding of AD, factors guiding its natural course are only insufficiently elucidated. We thus performed suction blistering in skin of adult patients with stable, spontaneous remission from previous moderate-to-severe AD during childhood. Samples were compared to healthy controls without personal or familial history of atopy, and to chronic, active AD lesions. Skin cells and tissue fluid obtained were used for single-cell RNA sequencing and proteomic multiplex assays, respectively. We found overall cell composition and proteomic profiles of spontaneously healed AD to be comparable to healthy control skin, without upregulation of typical AD activity markers (e.g., IL13, S100As, and KRT16). Among all cell types in spontaneously healed AD, melanocytes harbored the largest numbers of differentially expressed genes in comparison to healthy controls, with upregulation of potentially anti-inflammatory markers such as PLA2G7. Conventional T-cells also showed increases in regulatory markers, and a general skewing toward a more Th1-like phenotype. By contrast, gene expression of regulatory T-cells and keratinocytes was essentially indistinguishable from healthy skin. Melanocytes and conventional T-cells might thus contribute a specific regulatory milieu in spontaneously healed AD skin.

Keywords: atopic dermatitis; eczema; multiplex proteomics; single-cell RNA seq; spontaneous remission.

Figure 1

scRNA-seq map of skin cells from suction blister samples of spontaneously healed atopic dermatitis and healthy control skin. (A) UMAP of 7,269 cells integrated from spontaneously healed atopic dermatitis patients (n = 4) and healthy control individuals (n = 4) according to similarity of their transcriptome, resulting in 15 different color-coded clusters. (B) Unsupervised hierarchical clustering showing relatedness of cell clusters (average gene signatures; correlation distance metric and average linkage). (C–Q) Feature plots combining all samples of expression distribution for cluster-specific marker genes. Intensity of expression levels for each cell is color-coded (red) and overlaid onto UMAP plots. (R) Heat map displaying the top 10 differentially expressed genes (according to highest log-fold change ordered by smallest adjusted p-value using logistic regression with Bonferroni correction for each cluster compared with the rest of the dataset); upregulation is indicated in yellow, and downregulation in blue; gene names are shown on the left. (S,T) Color-coded frequencies of cell clusters for spontaneously healed atopic dermatitis (n = 1,849) and healthy control blister samples (n = 5,420) displayed as pie graphs. (U,V) Separate UMAP plots of spontaneously healed atopic dermatitis and healthy control blister samples. AD, atopic dermatitis; KC, Keratinocytes; MEL, Melanocytes; TC, T lymphocytes; LC, Langerhans cells; DC, Dendritic cells; TREG, regulatory T lymphocytes; UMAP, Uniform Manifold Approximation and Projection.

Figure 2

Melanocytes and T-cells show highest numbers of regulated genes in spontaneously healed atopic dermatitis in comparison to healthy control skin. (A) Number of differentially expressed genes (DEGs) within each cluster comparing spontaneously healed AD with healthy control samples, projected onto UMAP plots. Differential gene expression was defined as log fold change >|0.25| and adjusted p < 0.05 as calculated by logistic regression and Bonferroni correction. (B) Expression heat map of genes differentially expressed in melanocytes of spontaneously healed AD compared to healthy controls. Upregulation is indicated in yellow, downregulation in blue. Cell numbers in this heat map were down-sampled to depict equal numbers of cells between groups. (C) Volcano plot of differentially expressed genes in cells of the TC-1 cluster of spontaneously healed atopic dermatitis compared to healthy control samples depicted as log fold change (FCH), with a cut-off>|0.25|. Genes with a –log10 adjusted p > 1.3 (corresponding to an adjusted p < 0.05) are labeled in red. (D–I) Violin plots sorted per sample group within T-cell clusters showing distribution of normalized gene expression levels of the respective genes, as well as feature plots (right) combining all samples, with intensity of expression levels for each cell color-coded (red), overlaid onto UMAP plots. (J,K) Volcano plot of differentially expressed genes in cells of the TC-2 and the TC-5 cluster of spontaneously healed atopic dermatitis compared to healthy control samples depicted as log fold change (FCH), with a cut-off>|0.25|. Genes with a –log10 adjusted p > 1.3 (corresponding to an adjusted p < 0.05) are labeled in red. (L–Q) Violin plots of T-cell clusters showing distribution of normalized gene expression levels of the respective genes in healthy control (red) and spontaneously healed atopic dermatitis (green). AD, atopic dermatitis; DEGs, differentially expressed genes; HC, healthy control; SH, spontaneously healed; FCH, fold change; KC, Keratinocytes; MEL, Melanocytes; TC, T lymphocytes; LC, Langerhans cells; DC, Dendritic cells; TREG, regulatory T lymphocytes; UMAP, Uniform Manifold Approximation and Projection.

Figure 3

Multiplex proteomics identify markers of spontaneously healed AD. Volcano plots of proteins detected by proteomic multiplex assays in suction blister fluid, that are upregulated or downregulated in spontaneously healed atopic dermatitis (n = 4), depicted as log2 fold change (FCH) over active AD lesions (n = 4). Differences between groups were assessed using a linear mixed model (36) and Benjamini-Hochberg correction for multiple testing. Proteins with a –log10 adjusted p > 1 (corresponding to an adjusted p < 0.1) are labeled in red. AD, atopic dermatitis.

Figure 4

scRNA-seq map of skin cells from suction blister samples of spontaneously healed atopic dermatitis and active AD lesions. (A) UMAP of 14,443 cells integrated from spontaneously healed atopic dermatitis patients (n = 4) and active AD samples (n = 4) according to similarity of their transcriptome, resulting in 17 different color-coded clusters. (B,C) Number of differentially expressed genes (DEGs) within each cluster comparing spontaneously healed AD with active AD samples, separately for up- and downregulated DEGs, projected onto UMAP plots. Differential gene expression was defined as log fold change >|0.25| and adjusted p < 0.05 as calculated by logistic regression and Bonferroni correction. (D–F) Expression heat map of the top 20 upregulated genes in spontaneously healed AD compared to AD samples in the respective cell clusters. Upregulation is indicated in yellow, downregulation in blue. Cell numbers in heat maps were downsampled for better comparability of clusters. AD, Atopic dermatitis; KC, Keratinocytes; Mo/Ma, Monocytes/Macrophages; LC, Langerhans cells; TC, T lymphocytes; MEL, Melanocytes; NK, NK cells; TREG, regulatory T lymphocytes; DC, Dendritic cells; SH, Spontaneously healed; HC, healthy controls; UMAP, Uniform Manifold Approximation and Projection.