Large-scale analysis of longitudinal skin gene expression in systemic sclerosis reveals relationships of immune cell and fibroblast activity with skin thickness and a trend towards normalisation over time

Abstract

Objectives: Determine relationships between skin gene expression and systemic sclerosis (SSc) clinical disease features, and changes in skin gene expression over time.

Methods: A total of 339 forearm skin biopsies were obtained from 113 SSc patients and 44 matched healthy controls. 105 SSc patients had a second biopsy, and 76 had a third biopsy. Global gene expression profiling was performed, and differentially expressed genes and cell type-specific signatures in SSc were evaluated for relationships to modified Rodnan Skin Score (mRSS) and other clinical variables. Changes in skin gene expression over time were analysed by mixed effects models and principal component analysis. Immunohistochemical staining was performed to validate conclusions.

Results: Gene expression dysregulation was greater in SSc patients with affected skin than in those with unaffected skin. Immune cell and fibroblast signatures positively correlated with mRSS. High baseline immune cell and fibroblast signatures predicted higher mRSS over time, but were not independently predictive of longitudinal mRSS after adjustment for baseline mRSS. In early diffuse cutaneous SSc, immune cell and fibroblast signatures declined over time, and overall skin gene expression trended towards normalisation. On immunohistochemical staining, most early diffuse cutaneous SSc patients with high baseline T cell and macrophage numbers had declines in these numbers at follow-up.

Conclusions: Skin thickness in SSc is related to dysregulated immune cell and fibroblast gene expression. Skin gene expression changes over time in early diffuse SSc, with a tendency towards normalisation. These observations are relevant for understanding SSc pathogenesis and could inform treatment strategies and clinical trial design.

Keywords: autoimmune diseases; fibroblasts; scleroderma; systemic.

Figure 1:. Differentially expressed genes in systemic sclerosis initial biopsies compared to healthy controls.

(A) Heat map of differentially-expressed genes. (B) Top five overrepresented pathways (left), predicted upstream transcriptional regulators (middle), and predicted upstream cytokines and growth factors (right) in SSc compared to HC as determined by Ingenuity Pathway Analysis of differentially-expressed genes. (C) Numbers of differentially-expressed genes (FDR <0.05 in SAM analysis) in SSc patients with affected skin at the biopsy site compared to those with unaffected skin at the biopsy site and HCs (left) or between dcSSc, lcSSc, and HCs (right). (D) Principal component analysis of genes differentially-expressed by >1.5-fold or <0.67-fold in SSc compared to HC, highlighting controls, SSc with unaffected skin, and SSc with affected skin (left) or highlighting controls, limited cutaneous SSc, early diffuse cutaneous SSc (defined here as within three years of disease onset), or late diffuse cutaneous SSc (defined here as more than three years since disease onset). HC = healthy control, SSc = systemic sclerosis

Figure 2:. Cell type signatures in skin of SSc patients compared to healthy controls.

Cell type signature scores for each SSc baseline sample (n = 113). Scores represent the average fold-change (SSc/HC) for 125 cell type-specific signature genes (see Supplementary Methods). Bottom margin values indicate the percentage SSc biopsies with upregulated (red) and downregulated (blue) signatures compared to HCs. Patients were clustered based upon signature scores (average linkage, Euclidean distance). The blue boxes to the left of the cell type signature scores indicate the mRSS, and the purple/orange boxes indicate affected (skin score of 1, 2, or 3) vs unaffected (skin score of 0) skin at the site of the biopsy, with legends at the right of the figure. White boxes indicate no skin scores recorded at the time of the biopsy. SSc = systemic sclerosis, Ctrl = healthy control, mRSS = modified Rodnan skin score, KC = keratinocyte, Hair ORS = hair outer root sheet, DC = dendritic cell, NK cell = Natural Killer cell

Figure 3:. Comparison of dysregulated gene expression in follow up vs initial biopsies from early diffuse cutaneous SSc.

Principal component analysis of genes differentially-expressed by >1.5-fold or <0.67-fold in SSc compared to HC, highlighting controls and initial vs 3^rd biopsies from early dcSSc patients. p values for comparison between groups were determined as described in the supplementary methods. HC = healthy control, SSc = systemic sclerosis, Bx = biopsy, PC = principal component

Figure 4:. Immunohistochemical staining of CD3 and CD68 in initial and follow up skin biopsies from early diffuse cutaneous SSc.

Representative images of CD3 (A) and CD68 (B) in initial and follow up biopsies from an early dcSSc patient. (C) Flow diagram of initial and follow up skin biopsies from 17 early dcSSc patients, with red representing samples with CD3-positive cell counts equal to or greater than the baseline median value (High), and blue representing cell counts less than the baseline median value (Low). Each row represents one patient. (D) Same as (C), but for CD68-positive cell counts.