Fig. 1: Activation status of IRF5 in human SLE.

a Evaluation of IRF5 activation by nuclear translocation (NT). Fluorescent images of monocytes (Mo), stained with control IgG (left) or an anti-IRF5 antibody (middle and right) and then with a fluorochrome-conjugated secondary antibody (yellow), were captured by confocal microscopy. Nuclei were stained with DAPI (cyan). Representative images of cells with or without IRF5 NT, obtained from two of the SLE patients and one of the healthy control (HC) donors, respectively, analyzed in b are shown. Scale bars represent 5 μm. b IRF5 hyperactivation in SLE-associated monocytes. The IRF5 NT was defined as the nuclear/cytosolic IRF5 ratio of >1.5× most frequent ratio from the same sample containing 75–100 cells (see “Methods”). The proportion of monocytes featuring IRF5 NT in HC donors and SLE patients. c Correlation between IRF5 NT and ISG expression. A scatterplot of IRF5 NT prevalence among monocytes and the OAS1 mRNA level in peripheral blood are shown. d–f Persistence of aberrant IRF5 activation and ISG expression in remission-phase (RP)-SLE. Active-phase (AP)- and RP-SLE were defined as the SLEDAI-2K ≥ 5 and <5, respectively (d). The percentage of monocytes with IRF5 NT (e) and the OAS1 mRNA level in peripheral blood (f) in AP- and RP-SLE were analyzed. g Correlation between IRF5 NT and the anti-dsDNA antibody level in RP-SLE. A scatterplot of IRF5 NT prevalence among monocytes and serum anti-dsDNA antibody concentration in AP- and RP-SLE are presented. h, i Persistence of aberrant IRF5 activation and ISG expression after the standard therapy. Percentages of monocytes featuring IRF5 NT (h) and OAS1 mRNA levels in peripheral blood (i) from SLE patients treated or not treated with PSL were analyzed. j Effects of standard-of-care drugs on IRF5 activation. PBMCs from a HC donor were pretreated with 1.5 μM PSL, 3 μM HCQ, 60 μM mycophenolic acid (MPA), or 1 μM TPCA-1 (positive control) for 30 min and then stimulated with 3 μM R-848 for 60 or 120 min. The cell lysates were analyzed by a capillary-based immunoassay with antibodies against phosphorylated IRF5 (phospho-IRF5), total IRF5, and GAPDH as a loading control. Representative data from two independent experiments are depicted. HC: n = 25, SLE: n = 44 (b, c), AP-SLE: n = 27 (d, f), or 18 (e, g), RP-SLE: n = 31 (d, f) or 26 (e, g), PSL (−): n = 8 (h) or 13 (i), PSL (+): n = 36 (h) or 45 (i). Horizontal bars (b, d–f, h, i) represent median with interquartile range. Dashed lines (e, f, h, i) indicate the median of HC data. ***P < 0.001, ns: not significant (two-sided Mann–Whitney U test). Two-sided Spearman’s rank correlation coefficients (rs) and P value were used to assess the correlation (c, g).