PMC

Human PBMC were preincubated with chloroquine and transfected with indicated nucleic acids. IFN-α production was analyzed 24 hours after stimulation. Error bars represent s.d. A: The IFN-α-inducing activity of bacterial RNA (untreated or DNase treated) and DNA of L. monocytogenes, Staphylococcus aureus and Escherichia coli was analyzed. B: The IFN-α-inducing activity of RNAs from L. monocytogenes, L. ivanovii, E. coli, S. aureus and Acinetobacter baumannii, treated with DNase or calf intestine alkaline phosphatase (CIAP), were compared.

A: THP-1 and A549 cells were transfected with double stranded triphosphorylated RNA (3P-dsRNA), poly(dA-dT), bacterial DNA (bacDNA), plasmid DNA (pDNA) or double stranded 84 mer DNA oligonucleotides (dsODN); B: THP-1, A549, HepG2 and Colo205 cells were transfected with L. monocytogenes RNA, L. monocytogenes DNA or 3P-dsRNA. Type I IFN (THP-1, A549, Colo205) or CXCL10 (HepG2) production was analyzed 24 hours after stimulation. The relative induction of the indicated cytokine is depicted as percentage of induction by transfected L. monocytogenes (L.M.) RNA. C, D, E and F: THP-1, A549, HepG2 and Colo205 cells were infected with wt and hly- L. monocytogenes at the indicated MOI. Type I IFN (THP-1, A549, Colo205) or CXCL10 production (HepG2) was analyzed 24 hours after stimulation. Error bars represent s.d.

A: Murine BM-DCs were transfected with indicated stimuli. One out of two experiments is shown. Murine IFN-α secretion was analyzed 24 hours after stimulation. Error bars represent SEM. B: A549 cells were transfected with siRNA against RIG-I, MAVS or Luciferase (control). Cells were then infected with L. monocytogenes or transfected with L. monocytogenes RNA (L.m. RNA), L. monocytogenes DNA (L.m. RNA) or 3P-dsRNA 48 hours after knock-down. Type I IFN production was analyzed 24 hours after stimulation. B) THP-1 cells were electroporated with control siRNA or siRNAs against MAVS or STING. 72 hours after electroporation, THP-1 cells were infected with hly^- or wt L. monocytogenes at indicated MOI or transfected with plasmid DNA (pDNA) or RIG-I ligand (3P-dsRNA). Type I IFN production was analyzed 24 hours after stimulation. Relative type I IFN induction was normalized to cells transfected with siRNA against Luciferase and stimulated with pDNA. Error bars represent s.d.

THP-1, A549 and HepG2 were infected with FITC-tagged and EU-labeled wt and hly^- L. monocytogenes for the indicated duration. Cells were then fixed, stained with Alexa594-azide and counterstained with DAPI. Left column, wt L. monocytogenes infection 1 hr. Middle column, wt L. monocytogenes infection 4 hrs. Right column, hly^- L. monocytogenes infection 4 hrs. A: THP-1 cells. B: A549 cells. C: HepG2 cells. As determined by counting of single bacteria in cells (50 cells per slide were counted) the average bacterial load was 9(wt) and 4(hly^-) bacteria per cell for THP-1 cells, 6(wt) and 4(hly^-) bacteria per cell for A549 cells and 6(wt) and 5(hly^-) bacteria per cell for HepG2 cells, one representative experiment out of two is shown. Whole L. monocytogenes are labeled green with FITC and RNA is visible as red fluorescence (Alexa594), nuclei are stained by DAPI (blue).