Production of the pro-inflammatory cytokines IL-1α and IL-1β is independent of the five translocated protein synthesis inhibitors

<p>(A) B6 macrophages were infected with Δ5Δ<i>fla</i>-GFP+ and protein translation was measured between 2–6 hrs post infection by incorporation of the methionine analog AHA. Translation (incorporation of AHA) was compared between infected cells (GFP+, black line) and uninfected bystanders (GFP−, grey line). (B) B6 macrophages were infected with GFP expressing Dot+, Dot− and Δ5 strains of <i>L. pneumophila</i> and protein synthesis inhibition was compared between these strains by incorporation of AHA between 1–2 hrs (left graph) or 3–4 hrs (right graph). Graphs show translation in infected cells (GFP+). Cells incubated in the absence of the methionine analog (No AHA) were used as a negative control to show baseline staining. (C) WT and MyD88^−/− macrophages were infected with the indicated strains at MOI-15 and cytokine transcripts were analyzed at 6 hrs post infection by qRT-PCR. Data represent the mean fold induction and SEM of samples relative to uninfected controls. (D) ELISA measurement of IL-1α secretion from WT and MyD88^−/− macrophages infected with the indicated strains for 24 hrs. Data represent mean and SEM of triplicate samples. (E) Immunoblot analysis of pro-IL-1β in WT and MyD88^−/− macrophages challenged with the indicated <i>L. pneumophila</i> strains for 6 hrs. (F) B6 macrophages were challenged with <i>ΔflaA</i>-GFP or <i>Δ</i>5<i>Δfla</i> at MOI-15 for 6 hrs and sorted by Flow Cytometry. Pro-IL-1β levels were measured in both GFP+ and GFP− population by westernblot.