Fig 2: Transcriptome analysis by RNA-Seq defining the immunomodulatory activities of PW in THP-1-derived macrophages. (A) The heatmap of the top 79 differentially expressed genes (DEGs) among the four experimental groups: LPS stimulation (LPS), LPS with PW treatment (LPS-PW), PW treatment only (PW) and unstimulated control (Unstim); each with three biological replicates; top 57 DEGs of adjusted p-value < 0.05 and |log2 (fold change)| > 2 were identified for PW vs. Unstim group, and 22 DEGs with adjusted p-value < 0.05 and |log2 (fold change)| > 0.5 were shown for LPS-PW vs. LPS group. (B, C) Volcano plots showing all the gene expression pattern and highlighting the top 10 up- or down-regulated DEGs in PW vs. Unstim group (B) and LPS-PW vs. LPS group (C); the red, blue and gray color represents up-regulated, down-regulated and non-significant genes, respectively; the threshold line setting for adjusted p-value = 0.05 and |log2 (fold change)| = 1. (D, E) KEGG pathway enrichment analysis for PW vs. Unstim (D) and LPS-PW vs. LPS (E); all the pathways were significantly enriched with an adjusted p-value < 0.05; the red and blue color represents up-regulated and down-regulated pathways, respectively. (F G) Interaction analysis of pathways and related genes by ClueGO for PW vs. Unstim group (F) and LPS-PW vs. LPS group (G); the red and blue color represents up-regulated and down-regulated pathways and genes, respectively; the cut-off p-value = 0.05. (H, I) Immunoblotting confirming the inhibition of STAT1 activation (phosphorylation of STAT1, p-STAT1) at 2h (H) and IRF7 expression at 24 h (I) by PW under LPS stimulation; β-actin as the internal control. PW = 100 nM, LPS = 10 ng/mL, N ≥ 3.

Fig 3: Expression of interferon-related genes in the peripheral blood mononuclear cells of anti-MDA5+ idiopathic inflammatory myopathy (IIM) patients. (A) The expression levels of the nine validated genes were compared among anti-MDA5+ IIM patients (n = 29), anti-MDA5- IIM patients (n = 64), and healthy controls (n = 57). (B) Correlation analyses of anti-MDA5 antibody levels with the expressions of IFIH1, IRF7, and STAT1 genes in anti-MDA5+ IIM patients, with the correlation coefficient r values, p values, and 95% confidence intervals indicated. (C) Receiver operating characteristic analyses of the validated genes in distinguishing anti-MDA5+ patients. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 4: Validation of interferon-related genes in the monocytes of patients with idiopathic inflammatory myopathies (IIM). (A) Expression levels of DDX58, IFIH1, IRF7, STAT1, STAT2, ADAR, MNDA, TBX21, and TRIM25 detected in the monocytes of patients with IIM (n = 14) and healthy controls (HCs; n = 18) by qRT-PCR. (B) Expression of IRF7, p-IRF7, STAT1, p-STAT1, STAT2, p-STAT2, SHP1, and p-SHP1 detected in the monocytes of patients with IIM (n = 8) and HCs (n = 8) by Western blotting. (C) Receiver operating characteristic analyses of the genes expressed by monocytes in the diagnosis of IIM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 5: Validation of interferon-related genes in the peripheral blood mononuclear cells of patients with idiopathic inflammatory myopathies (IIM). (A) Expression levels of DDX58, IFIH1, IRF7, STAT1, STAT2, ADAR, MNDA, TBX21, and TRIM25 detected in patients with IIM (n = 93) and heathy controls (HCs; n = 57) by qRT-PCR. (B) Expression of IRF7, p-IRF7, STAT1, p-STAT1, STAT2, p- STAT2, SHP1, and p-SHP1 detected in patients with IIM (n = 5) and HCs (n = 8) by Western blotting. (C) Receiver operating characteristic analyses of the validated genes in the diagnosis of IIM. (D) Heat map of the correlation between laboratory parameters and gene expressions in patients with IIM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.