Cytokines are produced from both infected and bystander macrophages.

<p>(A) B6 macrophages were challenged with <i>ΔflaA</i>-GFP at MOI-15 for 4 hrs and sorted by Flow Cytometry. Cytokine and <i>Dusp</i> transcripts were measured in both GFP⁺ and GFP⁻ population by qRT-PCR. (B) B6 macrophages were infected with <i>ΔflaA</i>-GFP at MOI-15 for indicated times and levels of IL-1β were measured by Western blot. Bottom graphs indicate densitometry of IL-1β in GFP⁺ cells normalized to tubulin. (C) WT and MyD88^−/− macrophages were infected with indicated <i>L. pneumophila</i> strains at MOI-10 and intracellular cytokine levels were measured by flow cytometry. Top panels show TNF levels at 14 hrs post infection. To prevent secretion of TNF, cells were treated with Golgiplug (Brefeldin A) for 5 hrs before samples were collected. Bottom panels show intracellular IL-1α levels at 6 hrs post infection. Data shown are representative of at least 4 independent experiments. (D) Time course analysis of intracellular IL-1α levels in <i>ΔflaA</i>-GFP infected macrophages and (E) Comparison of intracellular IL-1α and DUSP1 levels in B6 macrophages infected with <i>ΔflaA</i>-GFP for 2 and 6 hrs. Infected and uninfected (bystander) cells were gated based on GFP signal and protein levels were compared to control macrophages that were left untreated. Red lines indicate GFP⁺ population; grey lines indicate GFP⁻ population and black lines show untreated macrophages.