Fig. 5: Suppression of IRF5 and type I IFN by a prototypical IRF5 inhibitor YE6144.

a Chemical structure of YE6144. b IRF5 NT in monocytes (Mo; left) or pDCs (right) sorted from human HC PBMCs that were pretreated with the vehicle (DMSO) or 1 μM YE6144 for 30 min and then were stimulated with 3 μM R-848 for 30 min. c IRF5 phosphorylation. Human HC PBMCs and mouse WT splenocytes were pretreated with either 1 and 3 μM YE6144, respectively, or DMSO for 30 min and then stimulated with 3 μM R-848 for 60 min. Cell lysates were analyzed by the capillary-based immunoassay with antibodies against phospho-IRF5, total IRF5, and GAPDH as a loading control. d NF-κB p65 NT in cells in b. e, f Type I IFNs. e Mouse WT splenocytes were pretreated with either DMSO or YE6144 for 30 min and next were stimulated with 2 μg/ml poly(U), 3 μM R-848, 1 μM CpG-A ODN, or 0.15 μM CpG-B ODN for the indicated period. Total RNA was isolated, and the expression of Ifnb1 and Ifna (detection of 12 subtypes) was analyzed by RT-qPCR. f Human HC PBMCs were pretreated with either DMSO (0) or YE6144 at the indicated doses for 30 min and then were stimulated with 3 μM R-848 for 24 h. IFN-β and IFN-α (detection of four subtypes) in culture supernatants was measured by ELISA. The concentration (0.03–10 μM) of YE6144 was plotted on a logarithmic scale. The red line represents a four-parameter log-logistic dose–response curve. Data in b, d were compiled from three independent experiments (n = 3 in total). Data in c, e, f are representative of two independent experiments (n = 3 in e and n = 3 [YE6144] or 6 [DMSO] in f for each experiment). Horizontal bars (b, d–f) denote mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (two-sided Student’s t test).