Fig 1: Dex regulates expression of ISGs both upstream and downstream of IFNβ in primary human macrophages. (A) Primary human monocyte-derived macrophages (n=7) were stimulated with 10 ng/ml LPS ± 100 nM Dex for 4 h, and IFNβ protein in supernatants was measured by Luminex assay. Right hand panel illustrates the same data with normalization to LPS alone. (B) Primary human monocyte-derived macrophages (n=7) were stimulated with 10 ng/ml LPS ± 100 nM Dex (left) or with 10 ng/ml IFNβ ± 100 nM Dex (right) for 4, 8 or 12 h. Rsad2 and Cxcl9 mRNAs were measured by qPCR, with normalization against untreated control for each individual macrophage culture. *, p < 0.05; **, p < 0.01; ***, p < 0.005; n.s., p > 0.05. C. Alveolar macrophages were isolated from histologically normal regions of lung resection tissue and treated with 1 μg/ml LPS ± 100 nM Dex for 12 h. IFNB1 and RSAD2 mRNAs were measured by qPCR, with normalization against untreated control for each individual sample. *, p < 0.05.
Fig 2: Dexamethasone inhibits expression of IFNβ in LPS-activated mouse macrophages via an unknown mechanism. (A) A RAW264.7 cell line stably transfected with an ISGF3-dependent SEAP (secreted alkaline phosphatase) reporter was treated with combinations of LPS (10 ng/ml), IFNβ (10 ng/ml), Ruxolitinib (Rux) and Dex as indicated for 24 h and reporter activity was assayed. The graph shows mean results from 3-6 independent experiments ± SEM, with normalization against reporter activity in the presence of LPS alone. *, p < 0.05 (Wilcoxon signed rank test); ****, p < 0.001 (ANOVA). (B) Mouse BMDMs were treated with combinations of LPS (10 ng/ml), IFNβ (10 ng/ml) and Dex (100 nM) as indicated. Cell lysates were generated and western blotted using antibodies against phosphorylated (Tyr 701) or total STAT1. Representative of three independent experiments. (C) Mouse BMDMs were stimulated with LPS for 4 h in the absence or presence of an IFNAR neutralizing antibody or isotype control (both 10 μg/ml). Cell lysates were prepared and western blotted as in (B) Representative of three independent experiments. (D) Mouse BMDMs were treated with LPS (10 ng/ml) or LPS + Dex (100nM) for the indicated times, and Ifnb1 mRNA was measured by qPCR. Mean of three independent experiments ± SEM. ***, p < 0.005. (E) Mouse BMDMs were treated with combinations of LPS (10 ng/ml) and Dex (100 nM) for 0.5 – 4 h, cell lysates were prepared and blotted for phosphorylated (activated) TBK1 or β-actin. Representative of two independent experiments. (F) Mouse BMDMs were treated as in E and lysates were blotted for phosphorylated (activated) IRF3 or β-actin.
Fig 3: DUSP1 contributes to Dex-mediated suppression of ISGs. (A) Dusp1+/+ or Dusp1-/- BMDMs (three independent isolates of each) were treated with LPS (10 ng/ml) for 4h with or without addition of Dex as indicated. IFNβ protein was measured by Luminex assay. The graph shows mean ± SEM, n = 3. ***, p < 0.005. (B) For each interferon-regulated gene of “antiviral” or “immmuno-modulatory” subsets (see Figures 2 , 3 ), Dex sensitivity was calculated as the ratio of expression in the presence of LPS + Dex vs. expression in the presence of LPS alone. In the plot of Dex sensitivity in Dusp1+/+ and Dusp1-/- BMDMs, the dotted diagonal represents the null hypothesis, that effects of Dex are independent of Dusp1 genotype. Selected outlier genes that do not fit the null hypothesis are highlighted. (C) Microarray-derived expression data for these outlier genes are illustrated. The pattern of expression of Ifnb1 itself is also shown. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.005; all in comparison to 4 h LPS treatment.
Fig 4: ATG5DD stabilization inhibits ISG expression via cell-intrinsic modulation of IFN-β expression and not through desensitization to IFNAR signalling.a, b ATG5DD cells, pretreated or not with Shield1 (Sh1), were treated with recombinant mouse IFN-β at the indicated concentration for 6, 10 or 20 h, followed by surface expression analysis of CD274 (a) and H-2Kb (b) by flow cytometry. c ATG5 DD cells were pretreated with anti-IFNAR1 antibodies followed by IAV infection. Ifnb1 expression was assayed by RT–qPCR. d In independent experiments, Ifnb1 induction was measured at 1.5 and 3.5 h post-infection in autophagy-competent (Sh1) vs. autophagy-null cells (∅). e, f ATG5DD cells, pretreated or not with Shield1 (Sh1), were infected with IAV for 1 h before monitoring IκBα degradation by immunoblot (e) and quantification with ImageJ (f). g ATG5DD cells transfected with NF-κB transcription activity GFP reporter for 20 h were pretreated or not with Shield1 (Sh1) and infected with IAV. Incucyte intra-incubator microscope allowed the monitoring of GFP-positive cells. a–d, f, g Graphs show mean and standard deviation of three (a–d, f) or four (g) biological replicates, and data are representative of three experiments. ns, not significant; *q < 0.05, **q < 0.01 (one-tailed unpaired t-test followed by Holm’s multiple testing correction)
Fig 5: E0771 tumor RNA-seq. A, Number of genes showing significant upregulation or down regulation at each of five time points of metronomic CPA treatment. A total of 2,633 genes met the thresholds for a significant response (fold-change > 2 at edgeR-adjusted P < 0.05) at one or more time points (Supplementary Table S5A). B, The set of 2,633 responsive genes was classified based on the time course of response, as detailed in Supplementary Table S5B. Each set was analyzed for overlap with the set of 188 genes that were up regulated by both 4HC and IFNB in cultured E0771 cells (Fig. 3B), and enrichment scores with significance by Fisher exact text calculated compared to a background set of all genes expressed at FPKM > 1, as shown in Supplementary Table S5B.
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