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. 2014 Sep 3;193(7):3600–3612. doi: 10.4049/jimmunol.1401088

FIGURE 1.

FIGURE 1.

Type I IFN regulates IL-10 production in M. tuberculosis–infected macrophages independently of IL-27 signaling. (A) WT macrophages were infected with M. tuberculosis in the presence of increasing concentrations of IFN-β, added at the time of infection, and levels of IL-10 in culture supernatant were determined by ELISA at 24 h postinfection. (B) WT macrophages were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of Il10 mRNA were determined by quantitative RT-PCR (qRT-PCR) at the time points indicated after infection. (C) WT and Ifnar1−/− macrophages were infected with M. tuberculosis and levels of IL-10 in culture supernatant were determined by ELISA at 24 h postinfection. (D) WT and Ifnar1−/− macrophages were infected with M. tuberculosis and levels of Il10 mRNA determined by qRT-PCR at the time points indicated after infection. (E) WT myeloid cells taken ex vivo from lungs (i) and BM (ii) were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of IL-10 in culture supernatant were determined by Luminex bead array at 24 h postinfection. (F) WT, Ifnar1−/−, and WT treated with IFN-β macrophages were infected with M. tuberculosis, and at 1 h postinfection ActD was added. mRNA was then taken at the time points indicated and Il10 mRNA levels were determined by qRT-PCR. (G) WT macrophages treated (or not) with 2 ng/ml IFN-β at the time of infection (i) and WT and Ifnar1−/− macrophages (ii) were infected with M. tuberculosis, and levels of IL-27 in culture supernatant were determined by ELISA at 24 h postinfection. (H and I) WT and Tccr−/− (IL-27Rα−/−) macrophages were infected with M. tuberculosis in the presence or absence of 2 ng/ml IFN-β, added at the time of infection, and levels of IL-10 (H) or TNF-α (I) in culture supernatant were determined by ELISA at 24 h postinfection. (J) WT and Tccr−/− (IL-27Rα−/−) macrophages were treated for 20 min with rIL-27 (50 ng/ml), rIFN-γ (10 ng/ml), or rIL-10 (10 ng/ml) and whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. (K) WT and Tccr−/− (IL-27Rα−/−) macrophages were stimulated for 0, 3, or 6 h with Pam3CSK4 (200 ng/ml) and then treated (or not) for 20 min with rIL-27 (50 ng/ml). WT macrophages treated with IFN-γ (10 ng/ml) or IL-10 (10 ng/ml) were included as positive controls for STAT-1 and STAT-3 phosphorylation, respectively. Whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. (L) WT and Tccr−/− splenocytes were treated for the indicated times with rIL-27 (50 ng/ml) or rIFN-γ (10 ng/ml). Whole-cell extracts were then analyzed by immunoblotting with the indicated Abs. Graphs show means ± SEM of triplicate samples, except for (E), which shows duplicates. For ELISA and Luminex bead array results, uninfected control samples were below the detection limit (20 and 5 pg/ml, respectively) for the cytokines measured (data not shown). Significance was determined using an unpaired t test (A, C, E, and G), a one-way ANOVA with a Bonferroni post hoc test (H and I), or a two-way ANOVA, with significance relative to WT (F). Data are representative of at least two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.