The Frustrated Host Response to Legionella pneumophila Is Bypassed by MyD88-Dependent Translation of Pro-inflammatory Cytokines
Figure 6
Production of the pro-inflammatory cytokines IL-1α and IL-1β is independent of the five translocated protein synthesis inhibitors
(A) B6 macrophages were infected with Δ5Δfla-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 L. pneumophila 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 L. pneumophila strains for 6 hrs. (F) B6 macrophages were challenged with ΔflaA-GFP or Δ5Δfla 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.