Association of Interferon- and transforming growth factor β-regulated genes and macrophage activation with systemic sclerosis-related progressive lung fibrosis

Abstract

Objective: Systemic sclerosis (SSc)-related interstitial lung disease (ILD) is one of the leading causes of mortality. We undertook this study to analyze the gene expression of lung tissue in a prospective cohort of patients with SSc-related ILD and to compare it with that in control lungs and with 2 prospective clinical parameters in order to understand the molecular pathways implicated in progressive lung disease.

Methods: Lung tissue was obtained by open lung biopsy in 28 consecutive patients with SSc-related ILD and in 4 controls. High-resolution computed tomography (HRCT) and pulmonary function testing (PFT) were performed at baseline and 2-3 years after treatment based on lung histologic classification. Microarray analysis was performed, and the results were correlated with changes in the HRCT score (FibMax) and PFT values. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry were used to confirm differential levels of messenger RNA and protein.

Results: Lung microarray data distinguished patients with SSc-related ILD from healthy controls. In the lungs of patients with SSc-related ILD who had nonspecific interstitial pneumonia (NSIP), expressed genes included macrophage markers, chemokines, collagen, and transforming growth factor β (TGFβ)- and interferon (IFN)-regulated genes. Expression of these genes correlated with progressive lung fibrosis defined by the change in FibMax. Immunohistochemistry confirmed increased markers of collagen (COL1A1), IFN (OAS1 and IFI44), and macrophages (CCL18 and CD163), and the positive correlation with the change in FibMax was confirmed by qPCR in a larger group of SSc patients with NSIP. Several genes correlated with both the change in FibMax (r > 0.4) and the change in % predicted forced vital capacity (r < -0.1), including IFN and macrophage markers, chemokines, and heat-shock proteins.

Conclusion: These results highlight major pathogenic pathways relevant to progressive pulmonary fibrosis in SSc-related ILD: macrophage emigration and activation, and up-regulated expression of TGFβ- and IFN-regulated genes.

Figure 1

A, Timeline of the study (see Patients and Methods). B, Heatmap showing the expression of genes clustered using complete linkage, hierarchical, unsupervised clustering. Expression values above the mean for each gene are indicated in red; those below the mean are indicated in green. The major dendrogram bifurcation places samples from all 4 healthy controls (represented by dark blue) onto the branch at the left along with 2 samples (from the middle and lower lobes) from 1 systemic sclerosis (SSc) patient with nonspecific interstitial pneumonia (NSIP) (represented by purple) (*). The right branch, including only samples from patients with SSc-related interstitial lung disease (ILD), bifurcates further into a middle sub-branch, which includes biopsy samples from both middle and lower lobes from 2 SSc patients with NSIP (represented by pink and light blue [**]) as well as single-lobe biopsy samples from 2 SSc patients with NSIP (represented by green [lower lobe] and orange [middle lobe]). In contrast, the sub-branch at the far right includes 3 of 6 samples from patients classified as having SSc with centrilobular fibrosis (1 sample represented by light green and 2 samples represented by red, where the patient represented by red had samples from both middle and lower lobes clustered together [***]). The other 3 samples on this branch (represented by black, green, and orange) were obtained from the lower lobes of SSc patients with NSIP. HRCT = high-resolution computed tomography; PFTs = pulmonary function tests; FibMax = sum of all scores in 6 lung zones; FVC = forced vital capacity.

Figure 2

Collagen and interferon (IFN) gene clusters highly expressed in SSc patients with NSIP. A and B, Heatmaps showing the expression of genes clustered using complete linkage, hierarchical, supervised clustering. Names of genes and Pearson correlation coefficients (r) are shown at the right of the collagen (A) and IFN (B) clusters. C–E, Messenger RNA expression of COL1A1 (C), IFI44 (D), and OAS1 (E) in samples obtained from lungs of healthy controls (HC) (n = 4) and SSc patients with NSIP (n = 25) using a larger population. Data are expressed as the fold change normalized to mRNA expression in a single sample from a healthy control. Symbols represent individual samples. Bars show the mean ± SD. F–H, Linear regression analysis of the relationship between the change in FibMax and lung mRNA expression of COL1A1 (r² = 0.39, P = 0.005) (F), IFI44 (r² = 0.36, P = 0.009) (G), and OAS1 (r² = 0.44, P = 0.007) (H). I, Cells staining positive for IFI44 (brown) in a healthy control lung. J and K, Strong staining for IFI44 (brown) within the interstitium and peribronchiolar areas of the lung of an SSc patient with NSIP. See Figure 1 for other definitions.

Figure 3

Macrophage gene cluster highly expressed in SSc patients with NSIP. A, Heatmap showing the expression of genes clustered using complete linkage, hierarchical, supervised clustering. Names of selected genes and Pearson correlation coefficients (r) are shown at the right. Blue brackets represent the position of genes described at right. B and C, Messenger RNA expression of CD163 (B) and CCL18 (C) in samples obtained from lungs of healthy controls (HC) (n = 4) and SSc patients with NSIP (n = 25) in a larger population. Data are expressed as the fold change normalized to mRNA expression in a single sample from a healthy control. Symbols represent individual samples. Bars show the mean ± SD. D and E, Linear regression analysis of the relationship between the change in FibMax and lung mRNA expression of CD163 (r² = 0.24, P = 0.03) (D) and CCL18 (r² = 0.20, P = 0.05) (E). F and G, Few cells staining positive for CCL18 (brown) in a healthy control lung. H–J, Cells staining positive for CCL18 (brown) within the interstitium and peribronchiolar areas of the lung of an SSc patient with NSIP. See Figure 1 for other definitions.