Skin gene expression correlates of severity of interstitial lung disease in systemic sclerosis

Abstract

Objective: We undertook this hypothesis-generating study to identify skin transcripts correlating with severity of interstitial lung disease (ILD) in systemic sclerosis (SSc).

Methods: Skin biopsy samples from 59 patients enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort or an open-label imatinib study (baseline visit) were examined by global gene expression analysis using Illumina HT-12 arrays. Skin transcripts correlating with concomitantly obtained forced vital capacity (FVC) values and the modified Rodnan skin thickness score (MRSS) were identified by quantitative trait analysis. Also, immunofluorescence staining for selected transcripts was performed in affected skin and lung tissue. Plasma levels of CCL2, soluble SELP, and soluble P-selectin glycoprotein ligand 1 (sPSGL-1) were examined in all patients enrolled in the GENISOS cohort (n = 266).

Results: Eighty-two skin transcripts correlated significantly with FVC. This gene list distinguished patients with more severe ILD (FVC <70% predicted) in unsupervised hierarchical clustering analysis (P < 0.001). These genes included SELP, CCL2, and matrix metalloproteinase 3, which are involved in extravasation and adhesion of inflammatory cells. Among the FVC correlates, 8 genes (CCL2, HAPLN3, GPR4, ADCYAP1, WARS, CDC25B, PLP1, and STXBP6) also correlated with the MRSS. Immunofluorescence staining revealed that SELP and CCL2 were also overexpressed in affected skin and lung tissue from SSc patients compared to those from controls. Plasma levels of CCL2 and sPSGL-1 correlated with concomitantly obtained FVC values (r = -0.22, P = 0.001 and r = 0.17, P = 0.015, respectively). This relationship was independent of potential confounders (age, sex, ethnicity, smoking status, anti-topoisomerase I positivity, treatment with immunosuppressive agents, MRSS, disease type, and disease duration).

Conclusion: A limited number of skin transcripts including genes involved in extravasation and adhesion of inflammatory cells correlate with severity of ILD.

Figure 1

Unsupervised hierarchical clustering of 82 skin transcripts that correlated with the concomitantly obtained forced vital capacity % predicted values. A, Only samples. B, Samples/genes. Samples are labeled according to study groups: those with interstitial lung disease (ILD) (yellow) and those with no ILD (purple).

Figure 2

Correlation of skin transcript levels of SELP (A) and CCL2 (B) with the concomitantly obtained forced vital capacity (FVC) % predicted value. The transcript levels have been log-transformed.

Figure 3

Left, Immunofluorescence staining with anti–P-selectin antibodies in skin (A) and lung tissue (B) from normal controls and patients with systemic sclerosis (SSc) or idiopathic pulmonary fibrosis (IPF). Arrows indicate double-positive cells. Original magnification × 400. SMA = α-smooth muscle actin. Right, Fluorescence intensity of P-selectin in the 3 groups. Bars show the mean ± SD.

Figure 4

Left, Immunofluorescence staining with anti-CCL2 antibodies in lung tissue from normal controls and patients with SSc or IPF. Arrows indicate double-positive cells. Original magnification × 400. Right, Fluorescence intensity of CCL2 in the 3 groups. Bars show the mean ± SD. See Figure 3 for definitions.

Figure 5

Correlations of plasma levels of CCL2 (A), soluble SELP (B), and soluble P-selectin glycoprotein ligand 1 (sPSGL-1) (C) with concomitantly obtained forced vital capacity (FVC) % predicted values.