Dissecting the heterogeneity of skin gene expression patterns in systemic sclerosis

Abstract

Objective: To examine the heterogeneity of global transcriptome patterns in systemic sclerosis (SSc) skin in a large sample of patients with SSc and control subjects.

Methods: Skin biopsy specimens obtained from 61 patients enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort and 36 unaffected control subjects with a similar demographic background were examined by Illumina HumanHT-12 bead arrays. Followup experiments using quantitative polymerase chain reaction and immunohistochemical analysis were also performed.

Results: We identified 2,754 differentially expressed transcripts in SSc patients compared with controls. Clustering analysis revealed 2 prominent transcriptomes in SSc patients: the keratin and fibroinflammatory signatures. Higher keratin transcript scores were associated with shorter disease duration and interstitial lung disease, while higher fibroinflammatory scores were associated with diffuse cutaneous involvement, a higher skin score at the biopsy site, and a higher modified Rodnan skin thickness score. A subgroup of patients with significantly longer disease duration had a normal-like transcript pattern. Analysis of cell type-specific signature scores revealed remarkable heterogeneity across patients. Significantly higher scores were calculated for fibroblasts (72% of patients), microvascular cells (61%), macrophages (54%), and dendritic cells (DCs) (49%). The majority of samples with significantly higher fibroblast scores (35 of 44 [80%]) had significantly increased macrophage and/or DC scores. Further analysis and immunohistochemical staining indicated that the keratin signature was not a general marker of keratinocyte activation but was in fact associated with an activation pattern in hair and adnexal structures.

Conclusion: Prominent fibroinflammatory and keratin signatures are present in SSc skin. Expression profiles of SSc skin show significant heterogeneity, and this finding might be useful for stratifying patients for targeted therapies or predicting the response to immunosuppression.

Figure 1

Numbers of differentially expressed (DE) genes in systemic sclerosis (SSc) patients with affected skin at the biopsy site compared with those with unaffected skin at the biopsy site and control subjects (A) and in patients with diffuse SSc compared with those with limited SSc and control subjects (B).

Figure 2

Unsupervised hierarchical clustering of skin samples based on the 2,754 transcripts that were differentially expressed in clinically affected systemic sclerosis (SSc) skin, unaffected SSc skin, and control skin. The orange bars and purple bars on the left indicate keratin and fibroinflammatory gene clusters, respectively.

Figure 3

Genes differentially expressed in systemic sclerosis (SSc) skin samples (versus control samples). Enrichment with genes specifically expressed in certain cell types is shown. Genes were ranked according to the degree to which they were specifically expressed in each of 14 cell types. Enrichment statistics quantify the degree to which genes that were significantly increased (A) or decreased (B) in SSc skin samples are among the genes specifically expressed with respect to each cell type. Positive enrichment statistics denote enrichment of differentially expressed genes with respect to genes most specifically expressed in a given cell type. Negative enrichment statistics denote enrichment of differentially expressed genes with respect to genes showing specifically low expression in a given cell type. ** = P < 0.001 by Wilcoxon’s rank sum test. DC = dendritic cell; KC = keratinocyte; ORS = outer root sheath; NK = natural killer.

Figure 4

Hierarchical clustering of patients with systemic sclerosis based on cell type signature scores. Each row represents a patient sample. Transcript scores for each patient were calculated with respect to 14 cell types. Scores were calculated based on fold-change estimates for 125 signature genes per cell type (expression in patient samples/average expression in 36 control samples). Triangles denote scores that are significantly high (▲) or low (▼) compared with all other human genes. Values in the 2 bottom rows are the percentages of patients with significantly high (red) or low (blue) scores. KC = keratinocyte; ORS = outer root sheath; NK = natural killer; DC = dendritic cell.

Figure 5

Representative images showing keratin 6 (KRT6), KRT16, and KRT85 staining of skin tissue from patients with systemic sclerosis (SSc) with a keratin transcript signature (n = 5) and unaffected control subjects (n = 5). Psoriatic skin was used as a positive control for KRT6 and KRT16. Arrows indicate hair follicles. Original magnification × 400.