A total of 154 patients with IIM including 129 patients with DM and 25 patients with immune-mediated necrotizing myopathy (IMNM) from China-Japan Friendship Hospital were enrolled in this study. DM was diagnosed based on the 2017 ACR/EULAR IIM criteria , and IMNM was diagnosed using ENMC IMNM criteria . Patients younger than 16 years of age and those exhibiting complications or other connective tissue diseases were excluded from the study. Additionally, we enrolled 30 healthy, age- and sex-matched volunteers as healthy controls (HCs). Informed consent was obtained from all participants. The Ethical Review Committee of the China-Japan Friendship Hospital (2019-25-K19) approved this study.
We collected the demographic features, clinical features, and laboratory data of the patients from electronic medical records. In the longitudinal study, 21 anti-MDA5-positive patients with DM were followed up for 1–42 months. The median follow-up duration was 22 months. We collected the blood samples during the follow-up period at each hospitalization. The interval between the two sample collections from a single patient was 1–25 months. ILD was confirmed by computed tomography and pulmonary function analyses . RP-ILD was diagnosed by a worsening radiologic interstitial status and the presence of respiratory symptoms such as progressive dyspnea and hypoxemia [29, 30]. For pulmonary function examination, the results of forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and diffusing capacity of carbon monoxide (DLco) were collected. The myositis disease activity was assessed by 10-cm visual analog scales (VAS) for muscle, six extramuscular organ systems including constitutional, cutaneous, joint, gastrointestinal, pulmonary, and cardiac, and the physician’s global assessment (PGA).
Detection of serum Gal-9 and MSA
An enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) was used to measure the serum Gal-9 levels. Additionally, MSAs were detected using an immunoblot assay kit (Euroimmun, Lübeck, Germany). Anti-3-hydroxy-3-methyl coenzyme A reductase protein autoantibodies were measured by ELISA (Inova Diagnostics Inc., San Diego, CA, USA). These assays were performed according to the manufacturer’s instructions.
Cell culture and treatment
- Peripheral blood mononuclear cells (PBMCs) culture and treatment
PBMCs were isolated by centrifugation on a Histopaque density gradient (Sigma-Aldrich, St. Louis, MO, USA). The isolated PBMCs were seeded into 96-well plates at 5 × 105 cells/mL in Roswell Park Memorial Institute 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 U/μg/mL penicillin/streptomycin (Gibco) at 37°C and 5% CO2 for 48 h. The supernatant was collected by centrifugation. An ELISA kit was used to determine Gal-9 levels in the supernatant, as described.
- MRC-5 fibroblasts culture and treatment
MRC-5 human lung fibroblasts were obtained from American Type Culture Collection (Manassas, VA, USA) and seeded into 6-well plates at 1 × 105 cells/mL in minimum essential medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Gibco), 1% non-essential amino acids (Gibco), and 100 U/μg/mL penicillin/streptomycin (Gibco) at 37°C and 5% CO2 for 8 h. Next, Gal-9 (Biolegend, San Diego, CA, USA) or transforming growth factor-β (TGF-β) (Peprotech, Rocky Hill, NJ, USA) were added. The proliferation of MRC-5 fibroblasts stimulated with Gal-9 for 24 or 48 h was tested with a luminescent cell viability assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Total RNA was isolated from PBMCs or cultured MRC-5 fibroblasts stimulated with Gal-9 for 24 h using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The mRNA levels were tested by SYBR-Green-based qRT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primers of target genes [Gal-9, Tim-3, CD44, MX1, IFIH1, monocyte chemoattractant protein-1(CCL2), interleukin 1β (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, tumor necrosis factor-α (TNF-α), IFNγ, CCL18, C-X-C motif chemokine ligand 4 (CXCL4), and CXCL10] are shown in Supplementary Table 1. The 2-ΔCt method was used to calculate the relative gene levels.
Lung sections were obtained from anti-MDA5-positive patients with DM. Diagnosis was obtained by surgical resection or percutaneous lung biopsy. Tissues were fixed in 10% formalin and embedded in paraffin, and subjected to antigen retrieval by heating and treatment with 3% hydrogen peroxide for 15 min. After incubation with rabbit anti-Gal-9 monoclonal antibody (1:500 dilution; Abcam, Cambridge, UK), anti-Tim-3 (1:400 dilution; Proteintech, Rocky Hill, NJ, USA), and anti-CD44 (1:50 dilution; Biolegend) overnight at 4°C, goat anti-rabbit IgG antibody (Gene Tech Shanghai Company Limited, Shanghai, China) was incubated with the tissue sections for 30 min at room temperature. 3,3′-Diaminobenzidine (Gene Tech Shanghai Company Limited) was used as a chromogenic reagent and hematoxylin was used for counterstaining.
Western blot analysis
MRC-5 fibroblasts were stimulated with Gal-9 or TGF-β for 48 h. Total protein from the cells was extracted by adding protein lysis buffer to the cells. Western blotting was conducted using primary antibodies of rabbit polyclonal anti-smooth muscle actin (SMA) (1:1000 dilution; Proteintech) and mouse monoclonal anti-GAPDH (1:1000 dilution; Abcam), followed by secondary antibodies including peroxidase-conjugated goat anti-mouse IgG (1:5000 dilution; Abcam) and peroxidase-conjugated goat anti-rabbit IgG (1:5000 dilution; Abcam). Enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA) was added to the membranes. Quantitative protein densitometry was performed with ImageJ software (NIH, Bethesda, MD, USA).
Data analysis was performed using GraphPad Prism V.7.01 (GraphPad, Inc., San Diego, CA, USA) and SPSS Version 22 (SPSS, Inc., Chicago, IL, USA). Numbers (percentages), mean ± standard deviation, or median values and interquartile range (IQR) were used to express the data. Student’s t-test or Mann–Whitney U-test was used for two-group comparisons. For comparison among multiple groups, the Kruskal–Wallis H-test was performed. The correlations of normally and non-normally distributed data were measured using Pearson’s correlation and Spearman’s correlation, respectively. Longitudinal data were analyzed with the generalized estimating equation model. p-Values below 0.05 were considered to indicate statistical significance.