Aberrantly Expressed Galectin-9 Is Involved in the Immunopathogenesis of Anti-MDA5-Positive Dermatomyositis-Associated Interstitial Lung Disease

Abstract

Background: Dermatomyositis (DM) associated rapidly progressive interstitial lung disease (RP-ILD) has high mortality rate and poor prognosis. Galectin-9 (Gal-9) plays multiple functions in immune regulation. We investigated Gal-9 expression in DM patients and its association with DM-ILD.

Methods: A total of 154 idiopathic inflammatory myopathy patients and 30 healthy controls were enrolled in the study. Cross-sectional and longitudinal studies were used to analyze the association between serum Gal-9 levels and clinical features. Enzyme-linked immunosorbent assay and qRT-PCR were used to examine Gal-9 expression in the sera and isolated peripheral blood mononuclear cells (PBMCs) from DM patients. Immunohistochemistry was performed to analyze the expression of Gal-9 and its ligand (T-cell immunoglobulin mucin (Tim)-3 and CD44) in lung tissues from anti-melanoma differentiation-associated gene 5 (MDA5)-positive patients. The effect of Gal-9 on human lung fibroblasts (MRC-5) was investigated in vitro.

Results: Serum Gal-9 levels were significantly higher in DM patients than in immune-mediated necrotizing myopathy patients and healthy controls (all p < 0.001). Higher serum Gal-9 levels were observed in anti-MDA5-positive DM patients than in anti-MDA5-negative DM patients [33.8 (21.9-44.7) vs. 16.2 (10.0-26.9) ng/mL, p < 0.001]. Among the anti-MDA5-positive DM patients, serum Gal-9 levels were associated with RP-ILD severity. Serum Gal-9 levels were significantly correlated with disease activity in anti-MDA5-positive DM patients in both cross-sectional and longitudinal studies. PBMCs isolated from anti-MDA5-positive DM patients (3.7 ± 2.3 ng/mL) produced higher levels of Gal-9 than those from immune-mediated necrotizing myopathy patients (1.1 ± 0.3 ng/mL, p = 0.022) and healthy controls (1.4 ± 1.2 ng/mL, p = 0.045). The mRNA levels of Gal-9 were positively correlated with the levels of type-I interferon-inducible genes MX1 (r = 0.659, p = 0.020) and IFIH1 (r = 0.787, p = 0.002) in PBMCs from anti-MDA5-positive DM patients. Immunohistochemistry revealed increased Gal-9 and Tim-3 expression in the lung tissues of patients with DM and RP-ILD. In vitro stimulation with Gal-9 protein increased CCL2 mRNA expression in MRC-5 fibroblasts.

Conclusions: Among anti-MDA5-positive DM patients, Gal-9 could be a promising biomarker for monitoring disease activity, particularly for RP-ILD severity. Aberrant expression of the Gal-9/Tim-3 axis may be involved in the immunopathogenesis of DM-ILD.

Keywords: biomarker; dermatomyositis; galectin-9; interstitial lung disease; melanoma differentiation-associated gene 5.

FIGURE 1

Serum galectin-9 (Gal-9) levels in patients with idiopathic inflammatory myopathies and healthy controls (HCs). (A) Patients with dermatomyositis (DM), patients with immune-mediated necrotizing myopathy (IMNM), and HCs. (B) Patients with DM divided into anti-melanoma differentiation-associated gene 5 (MDA5)-positive group and anti-MDA5-negative group. (C) Anti-MDA5-negative patients stratified by myositis-specific antibodies (MSAs). ARS: aminoacyl-tRN synthetase; TIF1-γ: transcriptional intermediary factor 1 γ; NXP-2: nuclear matrix protein 2; SAE: small ubiquitin-like modifier activating enzyme. Horizontal bars indicate median with interquartile range.

FIGURE 2

Correlations between serum galectin-9 (Gal-9) levels and clinical features in anti- melanoma differentiation-associated gene 5 (MDA5)-positive patients. (A) Serum levels of Gal-9 in patients with rapidly progressive interstitial lung disease (RP-ILD) and patients with non-RP-ILD. (B–D) Correlations between serum Gal-9 levels and pulmonary function impairment parameters. (B) Serum Gal-9 levels were correlated with forced vital capacity (FVC)% by Pearson’s correlation analysis. (C) Serum Gal-9 levels were not correlated with forced expiratory volume in 1s (FEV1)% by Spearman’s correlation analysis. (D) Serum Gal-9 levels were not correlated with diffusing capacity of carbon monoxide (DLco)% by Pearson’s correlation analysis. (E) Serum levels of Gal-9 in patients who survived and patients who died. (F,G) Correlations between serum Gal-9 levels and physician’s global assessment (PGA) visual analog scale (VAS) scores (F) and pulmonary VAS scores (G) by Spearman’s correlation analysis. (H) Serum levels of Gal-9 were correlated with ferritin levels by Spearman’s correlation analysis. (I) Serum levels of Gal-9 were correlated with lymphocyte counts by Spearman’s correlation analysis. (J) Longitudinal changes in serum levels of Gal-9 and PGA VAS scores over time in 21 anti-MDA5-positive patients. The serum levels of Gal-9 were significantly correlated with PGA VAS scores using the generalized estimating equation model. Horizontal bars indicate median with interquartile range.

FIGURE 3

Concentrations of galectin-9 (Gal-9) in peripheral blood mononuclear cells (PBMCs). PBMCs derived from patients with idiopathic inflammatory myopathies and healthy controls (HCs). (A) Gal-9 levels in PBMCs derived from patients with dermatomyositis (DM), patients with immune-mediated necrotizing myopathy (IMNM), and HCs. (B,C) mRNA expression of type-I interferon-inducible gene myxovirus resistance 1 (MX1) (B) and interferon induced with helicase C domain 1 (IFIH1) (C) in patients with DM, patients with IMNM, and HCs. (D,E) Correlations between Gal-9 and MX1 (D) and IFIH1 (E) mRNA expression levels in patients with DM. Horizontal bars indicate mean ± standard deviation.

FIGURE 4

Immunohistochemical staining of galectin-9 (Gal-9) in lung sections. (A–F) Surviving 39-year-old female patient with anti-melanoma differentiation-associated gene 5 (MDA5)-positive dermatomyositis (DM) with no interstitial lung disease (ILD). (G–L) Surviving 27-year-old male patient with anti-MDA5-positive DM with non-RP-ILD. (M–R) Non-surviving 55-year-old male patient with anti-MDA5-positive DM with RP-ILD. (A,B,G,H,M,N) Staining with anti-Gal-9 antibody. (C,D,I,J,O,P) Staining with anti-T-cell immunoglobulin mucin (Tim)-3 antibody. (E,F,K,L,Q,R) Staining with anti-CD44 antibody. The red arrows indicate Gal-9-expressing macrophages. The green arrows indicate Tim-3-expressing alveolar epithelial cells.

FIGURE 5

Effects of galectin-9 (Gal-9) on the function of MRC-5 fibroblasts. (A) Monocyte chemoattractant protein-1 (CCL2) mRNA levels in Gal-9-treated MRC-5 fibroblasts compared to the control group. (B) Proliferation of MRC-5 fibroblasts stimulated with Gal-9 for 24 h. (C) α-smooth muscle actin (SMA) protein expression of MRC-5 fibroblasts treated with transforming growth factor-β (TGF-β) or Gal-9. The graphs show the mean ± standard deviation of and each graph determined from at least three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.