Background/Purpose: Skin gene expression profiling can distinguish SSc from normal skin and can detect different subsets of disease. Previous studies have reported a cross-sectional relationship between the expression of genes in whole skin and the modified Rodnan skin score (mRSS). We aim to compare gene expression in early versus late dcSSc and define cells responsible for the upregulation of candidate genes.

Methods: Total RNA was extracted from forearm skin biopsies of 3 early dcSSc anti-RNA polymerase III positive (ARA+) patients and 3 late dcSSc ARA+ patients. COMP, chemokine ligand 2 (CCL2), PDGF-A, collagen type I a 1 chain (COL1A1), interferon Induced Protein 44 (IFI44), IL-6, MMP3, SERPINE, TGF-b and thrombospondin 1 (THBS1) was measured using quantitative PCR analysis. mRSS was assessed in all patients and correlated with normalized gene expression. RNA in situ hybridization for a-actin-2 (ACTA2), CCL2, COL1A1, COL3A1, COMP, SERPINE and THBS1 mRNA was performed using RNAscope¨ 2.5 LS Reagent Kit in early dcSSc ARA+ patients (n=2) and healthy controls (HC) (n=3). Semi-quantitative scoring criteria were applied (score from 0 to 4).

Results: Whole skin relative normalized gene expression of COMP, CCL2, IL-6, SERPINE and THBS1 was significantly (p < 0.05) higher in early compared to late SSc skin (COMP mean 25049.15 ± 10862.16 vs 5256.47 ± 2380.53; CCL2 mean 1722.04 ± 598.93 vs 638.25 ± 279.96; IL-6 mean 43.98 ± 40.76 vs 11.91 ± 5.44; SERPINE mean 1770.32 ± 704.46 vs 184.48 ± 61.37; THBS1 mean 7190.75 ± 309.89 vs 1176.81 ± 235.35) [Figure 1]. There was a positive correlation between SERPINE and THBS1 and mRSS (SERPINE r² = 0.493, p = 0.09; THBS1 r² = 0.91, p = 0.02). Whole skin relative normalized gene expression of all the remaining genes, except for IFI44, was also higher in early compared to late patients although not significantly.  RNAscope analysis revealed positive SERPINE staining in subsets of fibroblast-like cells located in the mid-dermis in early dcSSc only (score 3 and 4 in early dcSSc vs score 0 in HCs). An increased number of THBS1+ and COMP+ fibroblast-like cells (α-smooth muscle actin +)  were found in the deep dermis of early dcSSc (score 4 in early dcSSc pts vs score 0, 2 and 3 in HCs).

Conclusion: SERPINE, THBS1, COMP, CCL2 and IL-6 are upregulated in early compared to late dcSSc patients. THBS1 and COMP are key genes associated with skin involvement and both are TGF-b regulated. RNAscope technology not only confirmed the upregulation of SERPINE, COMP and THBS1 in early dcSSc, but detected the expression of these genes in the dermis of patients in subsets of cells. These findings highlight how specific cells may be responsible for the pro-fibrotic and inflammatory changes typical of the early stages of the disease. Upregulation of fundamental pro-fibrotic and inflammatory genes could be crucial in the early stages of SSc and specific subsets of cells in the dermis could be the responsible for this.