Extended Data Fig. 5: IL-10 suppresses expression of IRF1 and IRF5 which mediate IFNB induction in human monocytes.
a, Immunoblot of IRF1 (top panel) and IRF5 (bottom panel) using whole cell lysates of monocytes pretreated with IL-10 followed by the indicated time course of LPS stimulation. Representative blots from 3 independent donors are depicted, HPRT is used as loading control. b, mRNA of indicated genes was measured by RT-qPCR and normalized relative to GAPDH mRNA in cells stimulated with IL-10 for 18 h and then challenged with LPS for 3 h under two conditions. No washout of IL-10 (labeled 0): IL-10 remained in the culture during the LPS challenge; washout of IL-10 (labeled 24): IL-10 was removed and the cells were cultured for an additional 24 h before being challenged with LPS for 3 h. (n = 3 independent donors). Data are depicted as mean ± SEM. **p < 0.001; ***p ≤ 0.0005; ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. c, IGV gene tracks of IL-10-induced STAT3 binding at IRF1 regulatory elements. d, IFNB mRNA was measured by qPCR and normalized relative to GAPDH mRNA in cells stimulated IL-10 for 18 h and challenged with LPS at various time points (n = 3 independent donors). Data are depicted as mean ± SEM. ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. e, mRNA of indicated genes was measured by RT-qPCR and normalized relative to GAPDH. Cells were treated with IL-10 for 18 h and stimulated with exogenous IFN-β to assess whether IL-10 blocks induction of ISGs (n = 6 independent donors). Data are depicted as mean ± SEM. *p < 0.05; ***p ≤ 0.0005; ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. f, h Heatmaps of z-score normalized expression of f, TNF RNA-seq showing regulation of IRF and ISG genes by IL-10, h, of LPS RNA-seq showing regulation of IRFs and ISGs by IL-10. g, Immunoblot of IRF1 and p38 using whole cell lysates from IRF1 or HPRT-edited cells (CRIPSR-Cas9 mediated edits) and stimulated with LPS for 3 h (representative blot from one out of 3 independent donors). Created in https://BioRender.com.
