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. Author manuscript; available in PMC: 2019 Mar 22.
Published in final edited form as: Sci Signal. 2017 Dec 12;10(509):eaan2392. doi: 10.1126/scisignal.aan2392

Fig. 5. Impaired IRF8 induction compromises amplification of the IFN response in older monocytes, and restoring IRF normalizes this response.

Fig. 5.

Monocytes from younger (n=19) and older (n=12) human donors were (A) transfected with a RIG-I–specific ligand or (B) infected with PR8 IAV, and IRF8 expression was measured by qPCR at the indicated timepoints. Expression values for each donor were normalized to HPRT. (C, D) Younger (n=4) and older (n=3) monocytes were transfected with a RIG-I–specific ligand for 6 hours, IRF8 protein abundance was measured by flow cytometry (C), and mean fluorescence intensity (MFI) values (D) were quantified. Data is representative of two independent experiments. (E) Monocytes from 5 younger donors were left untreated or were treated with cyclohexamide (CHX) for 4 hours prior to mock transfection or RIG-I stimulation for 6 hours, after which IRF8 expression was quantified by qPCR. (F, G) Monocytes from younger donors were treated with an IRF8-specific siRNA or RNA-induced silencing complex (RISC)-free control for 48 hours, then stimulated with a RIG-I–specific ligand. IRF8 expression was quantified by qPCR after 6 hours of RIG-I stimulation (F), and IFN-β secreted into the supernatant was quantified by ELISA after 12 hours of RIG-I stimulation. Data is representative of two independent experiments. (H, I) Monocytes from older donors (n=16) were transfected with an IRF8 or control lentiviral (LV) construct for 48 hours, then stimulated with a RIG-I–specific ligand. IRF8 expression was quantified by qPCR after 6 hours of RIG-I stimulation (H), and secreted IFN-β was measured by ELISA after 12 hours of RIG-I stimulation (I). (J) Monocytes from younger (n=7) and older (n=7) donors were transfected with a control or IRF8 lentiviral construct for 48 hours, stimulated with a RIG-I–specific ligand for 6 hours, and IRF7 MFI was quantified by flow cytometry. Data are means ± SEM. * P<0.05,** P<0.01, ***P<0.001; Unpaired or Paired Student t-test or Two-way Repeated Measures ANOVA.