Interferon and alternative activation of monocyte/macrophages in systemic sclerosis-associated pulmonary arterial hypertension

Abstract

Objective: To explore the relationship between biomarkers of pulmonary arterial hypertension (PAH), interferon (IFN)-regulated gene expression, and the alternative activation pathway in systemic sclerosis (SSc).

Methods: Peripheral blood mononuclear cells (PBMCs) were purified from healthy controls, patients with idiopathic PAH, and SSc patients (classified as having diffuse cutaneous SSc, limited cutaneous SSc [lcSSc] without PAH, and lcSSc with PAH). IFN-regulated and "PAH biomarker" genes were compared after supervised hierarchical clustering. Messenger RNA levels of selected IFN-regulated genes (Siglec1 and MX1), biomarker genes (IL13RA1, CCR1, and JAK2), and the alternative activation marker gene (MRC1) were analyzed on PBMCs and on CD14- and CD14+ cell populations. Interleukin-13 (IL-13) and IL-4 concentrations were measured in plasma by immunoassay. CD14, MRC1, and IL13RA1 surface expression was analyzed by flow cytometry.

Results: Increased PBMC expression of both IFN-regulated and biomarker genes distinguished SSc patients from healthy controls. Expression of genes in the biomarker cluster, but not in the IFN-regulated cluster, distinguished lcSSc with PAH from lcSSc without PAH. The genes CCR1 (P<0.001) and JAK2 (P<0.001) were expressed more highly in lcSSc patients with PAH compared with controls and mainly by CD14+ cells. MRC1 expression was increased exclusively in lcSSc patients with PAH (P<0.001) and correlated strongly with pulmonary artery pressure (r=0.52, P=0.03) and higher mortality (P=0.02). MRC1 expression was higher in CD14+ cells and was greatly increased by stimulation with IL-13. IL-13 concentrations in plasma were most highly increased in lcSSc patients with PAH (P<0.001).

Conclusion: IFN-regulated and biomarker genes represent distinct, although related, clusters in lcSSc patients with PAH. MRC1, a marker for the effect of IL-13 on alternative monocyte/macrophage activation, is associated with this severe complication and is related to mortality.

Figure 1

Biomarker and interferon (IFN)–regulated gene expression in limited cutaneous systemic sclerosis (lcSSc). A, Heat map showing the expression of genes clustered using average linkage, hierarchical, and supervised clustering. Individual subjects are listed at the top. Top right, Cluster of IFN-regulated genes; middle right, cluster of biomarker genes; bottom right, JAK2 cluster. Values above the mean expression level of each gene are indicated in red; those below the mean expression level are indicated in green. B, Relative expression of all genes in each cluster in A. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the minimum and maximum values. Lines inside the boxes represent the mean. lSSc-NoPAH = lcSSc without pulmonary arterial hypertension; lSSc-PAH = lcSSc with PAH.

Figure 2

IFN-regulated gene expression in lcSSc and diffuse cutaneous SSc (dcSSc, or dSSc). a and c, Expression of Siglec1 (a) and MX1 (c) in peripheral blood mononuclear cells (PBMCs) from healthy controls (HC), dcSSc patients, lcSSc patients without PAH, lcSSc patients with PAH, and patients with idiopathic PAH (IPAH). b and d, Expression of Siglec1 (b) and MX1 (d) in PBMC, CD14−, and CD14+ populations in lcSSc patients without PAH (n = 4), lcSSc patients with PAH (n = 5), and healthy controls (n = 3) (lcSSc patients without PAH and lcSSc patients with PAH are combined). Data are expressed as the fold change normalized to mRNA expression in a single sample from a healthy control subject. Bars show the mean ± SEM. # and ## = P < 0.05 versus all SSc patients. See Figure 1 for other definitions.

Figure 3

PAH biomarker gene expression in diffuse cutaneous SSc (dcSSc, or dSSc) and lcSSc. a, c, and e, Expression of IL13RA1 (a), CCR1 (c), and JAK2 (e) in peripheral blood mononuclear cells (PBMCs) from healthy controls (HC), dcSSc patients, lcSSc patients without PAH, lcSSc patients with PAH, and patients with idiopathic PAH (IPAH). b, d, and f, Expression of IL13RA1 (b), CCR1 (d), and JAK2 (f) in PBMC, CD14−, and CD14+ populations in lcSSc patients without PAH (n = 4), lcSSc patients with PAH (n = 5), and healthy controls (n = 3) (lcSSc patients without PAH and lcSSc patients with PAH are combined). Data are expressed as the fold change normalized to mRNA expression in a single sample from a healthy control. Bars show the mean ± SEM. # = P < 0.001, by analysis of variance. See Figure 1 for other definitions.

Figure 4

MRC1 expression in SSc patient populations and MRC1 expression by monocytes. a, Expression of MCR1 in peripheral blood mononuclear cells (PBMCs) from healthy controls (HC), patients with diffuse cutaneous SSc (dcSSc, or dSSc), lcSSc patients without PAH, lcSSc patients with PAH, and patients with idiopathic PAH (IPAH). Shaded circles represent lcSSc patients with PAH who died during the 2 years of followup in this study. b, Linear regression analysis of the relationship between the mean pulmonary artery pressure (PAP) by right-heart catheterization and the expression of MRC1 in PBMCs from lcSSc patients with PAH. c, Expression of CD14 on PBMC, CD14−, and CD14+ populations from SSc patients and controls. d, Expression of MRC1 on PBMC, CD14−, and CD14+ populations in lcSSc patients without PAH (n = 4), lcSSc patients with PAH (n = 5), and healthy controls (n = 3) (lcSSc patients without PAH and lcSSc patients with PAH are combined). e, Expression of Siglec1, MRC1, and IL13RA1 on PBMCs from healthy controls (n = 7) after 18 hours of stimulation with 20 ng/ml interleukin-13 (IL-13) or without IL-13 stimulation (control). f, Plasma IL-13 concentration in healthy controls, dcSSc patients, lcSSc patients without PAH, and lcSSc patients with PAH. Data in a and c-e are expressed as the fold change normalized to mRNA expression in a single sample from a healthy control. Bars show the mean ± SEM. # = P < 0.001, by analysis of variance. ELISA = enzyme-linked immunosorbent assay (see Figure 1 for other definitions).

Figure 5

MRC1 (CD206) surface expression on peripheral blood mononuclear cells (PBMCs) from lcSSc patients with PAH. A, Double staining (CD14+CD206+) of PBMCs before (4.81%) and after (12.8%) stimulation with interleukin-13 (IL-13). B, Induction of IL13RA1 and CD206 surface expression on all PBMCs before (red lines) and after (blue lines) 3 hours of stimulation with 20 ng/ml IL-13. After IL-13 stimulation, positive cell expression of IL13RA1 changed from 51.5% (red) to 53.5% (blue), and positive cell expression of CD206 changed from 8.45% (red) to 15.3% (blue). Horizontal bars represent stained cells. C, Expression of IL13RA1 MRC1 and IL13RA1 mRNA before and after IL-13 stimulation. D, MRC1 mRNA expression on healthy PBMCs stimulated for 24 hours with IL-13 or with several Toll-like receptor (TLR) ligands. Data are expressed as the fold change normalized to mRNA expression in a single sample from a healthy control. See Figure 1 for other definitions.