Induction of type I IFN genes.

<p>Samples derived from two biological replicates per time point were used for subsequent GeneChip probe arrays. (A) Heat map of the time course of IRG expression in DCs after exposure to <i>Lm</i>. (B) Heat map of the time courses of IFNβ and IFNα gene expression. RNAs were collected at different time points and the gene expression levels were measured by microarray analysis. Heat maps were generated with the GeneSpring hierarchical clustering algorithm. Data show mean fold-changes normalized to the pre-exposure 0 hr time point.</p

IFNγ production by NK cells cultured with BMDCs stimulated with <i>Lm</i> or <i>E. coli</i>.

<p>BMDCs were infected with <i>Lm</i> or <i>E. coli</i> at an MOI of 20 and cultured with syngeneic NK cells for 18 hr. Where indicated, recombinant IFNβ was added to the co-culture one hour after infection. Levels of IFNγ in the culture supernatants were quantified by ELISA. (A) Experimental design. (B) Co-culture experiments. The means ± SDs of three independent experiments are shown. p value <0.01.</p

Type I IFN production by <i>Lm</i>-infected DCs.

<p>(A) D1 cells were infected with <i>Lm</i> at an MOI of 40. Supernatants were collected at different time points and IFNβ and IFNα levels were quantified by ELISA. (B) D1 cells were infected with <i>Lm</i> at an MOI of 40. RNA was extracted at different time points and used for qRT-PCR analysis. The fold-increases, relative to β-actin, for IFNα4, IFNα9, IFNα5, IFNα2 are shown. (C) D1 cells were infected with <i>Lm</i>, and the RNA was extracted and analyzed by RT-PCR. IFNα6 and IFNα1 mRNA levels are shown. Data shown are representative of at least three independent experiments.</p

<i>In vivo</i> IFNβ production during <i>Lm</i> infection.

<p>Mice (n = 5/group) were injected with 1×10⁶ CFU of <i>Lm</i> and <i>E. coli</i>. RNA was extracted at 4 hr, 8 hr and 24 hr p.i. and the IFNβ mRNA was quantified. (A) IFNβ gene expression from total spleen at the time points indicated. (B) IFNβ gene expression in CD11c⁺ and CD11c⁻ cells purified from total spleen. (C) IFNβ gene expression in total spleen from mice infected with <i>E. coli</i> (1×10⁶ CFU) as a positive control. The housekeeping gene <i>PPIA</i> was used as a reference to normalize data. Data shown are representative of at least three independent experiments.</p

Type I IFN production by BMDCs.

<p>BMDCs from C57BL/6 mice were activated with <i>Lm</i> or <i>E. coli</i> at an MOI of 20. (A) IFNβ production by BMDCs. (B) IFNα production at the time points indicated was evaluated by ELISA. Data shown are representative of at least three independent experiments.</p

NK cell activation in the spleen of mice infected with <i>Lm</i>.

<p>Mice (n = 5/group) were infected with <i>Lm</i> (1×10⁶ CFU)± IFNβ. Spleens were removed five hours after infection and NK cell activation was evaluated. (A) Intracellular staining for IFNγ in DX5-positive cells. (B) Splenocytes were co-cultured with YAC-1 cells and the percentage of target cell lysis was determined after three hours; p-value <0.01. The mean of three independent experiments is shown.</p

Survival assay of mice infected with <i>Lm</i>.

<p>Mice (n = 5/group) were injected with a lethal dose of <i>Lm</i> alone (1×10⁶ CFU) or with IFNβ (38,000 U). Survival was monitored daily, for up to 18 days. Data shown are representative of at least three independent experiments. All experiments were performed using protocols approved by University of Milano-Bicocca Animal Care and Use Committee. Mice were housed in containment facilities of the animal facility and maintained on a regular 12∶12 hour light:dark cycle with food and water ad libitum.</p

Bacterial burden in the spleen of mice infected with <i>Lm</i>.

<p>Bacterial burden was measured in five mice per group. Mice were infected with sub-lethal doses (5×10⁴ CFU) of <i>Lm</i> ± IFNβ and killed after five hours. <i>Lm</i> titers were determined in the spleen and expressed as CFUs. Recombinant IFNβ was given one hour post-infection, as indicated; p = 0.0001. The mean of three independent experiments is shown.</p