M5 protein-dependent type I IFN-signaling is required for secretion of IL-10.

<p><b>A)</b> B6 macrophages were infected with M5 or ΔM5 bacteria, as indicated. Macrophages were lysed at the indicated time points (hours) post infection and analyzed for activation of Stat1 and Stat2 by Western blot. Uninfected (UI) macrophages were analyzed as control. Activated (<i>i</i>.<i>e</i>. phosphorylated) transcription factors were detected with phospho-specific primary antibodies (indicated with p). Shown is one experiment representative of three. <b>B)</b> B6 macrophages were infected with M5 or ΔM5 for 4 hours, and IFNβ transcripts were measured by RTqPCR. mRNA levels are presented as fold-change relative to UI control macrophages. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of three independent experiments. <b>C)</b> Kinetic analysis of IFNβ secretion from B6 macrophages infected as indicated. Results shown (mean; <i>n</i> = 2 per group) are representative of two independent experiments. <b>D)</b> B6 and IFNAR-KO macrophages were infected with M5 or ΔM5, or UI, as indicated. Culture supernatants were collected 24 hpi and assayed for indicated cytokines. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of three independent experiments. <b>E)</b> B6 macrophages were infected with wild type M5 bacteria in the presence of titrated amounts (final concentration indicated) of a neutralizing anti-mouse IFNAR mAb or mouse IgG1 isotype control, as indicated. UI macrophages treated with the anti-IFNAR mAb were analyzed as control. Cytokines were assayed 24 hpi. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of two independent experiments. <b>F)</b> B6 and IFNAR-KO macrophages were infected with <i>S</i>. <i>pyogenes</i> strains of six different serotypes (two that are serum opacity factor positive [OF⁺], and four that are OF^-), as indicated. Cytokines were assayed 24 hpi. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of two independent experiments. ANOVA (*<0.033; **<0.002; ***<0.001).