Overview of pattern recognition receptors and corresponding ligands used in this project.

<p>Overview of pattern recognition receptors and corresponding ligands used in this project.</p

Hirudin-WBA – application as fast screening method to determine immunostimulatory side effects of siRNA therapeutics.

<p>a–b – PBMC were stimulated with siRNA without (no meth) or with three 2′-O ribose methylated nucleotides (3x meth) delivered with p-L-Arginine (pArg) in increasing concentrations as indicated. After 48 h concentrations of IFN-α (a) and TNF-α (b) in supernatant was analyzed by ELISA. c–d – Done as described for (a) and (b) but whole blood assay anticoagulated with Hirudin was used. Results of eight donors are shown as mean +/− SD in (a–d). For sequence details of siRNA used see lower right paragraph.</p

RNA ligands for TLR7 and RIG-I induce IFN-α in Hirudin-whole blood assay.

<p>a – Whole blood was anticoagulated with Heparin, EDTA or Hirudin and stimulated with 0.8 µg/ml 3p-dsRNA, 9.2s-RNA or A20-RNA (negative control) delivered with Lipofectamine2000 or DOTAP. After 48 h supernatant was analyzed for IFN-α induction by ELISA. Graph shows percentage of induced IFN-α compared to 3p-dsRNA and 9.2s-RNA respectively (induced IFN-α concentrations: 3p-dsRNA: 2641 pg/ml; 9.2s-RNA: 1438 pg/ml). Results of four donors are shown as mean +/− SD. b – 3p-dsRNA was incubated with Lipofectamine2000 and 9.2s-RNA with DOTAP with or without addition of Heparin or Hirudin. Complexes were digested by RNase and subsequently analyzed by gel electrophoresis. c – Whole blood of three different donors anticoagulated with Hirudin was stimulated at two different time points with 0.8 µg/ml 3p-dsRNA, 9.2s-RNA or A20-RNA (negative control). After 48 h supernatant was analyzed for IFN-α using ELISA. Mean of duplicates +/− SD are shown for each timepoint. d – Whole blood was anticoagulated with Hirudin and stimulated with increasing concentrations of 3p-dsRNA and 9.2s-RNA. Results of eight donors are shown as mean +/− SD. e – Done as described for (d) but Protamine (Prot) and poly-L-Arginine (pArg) were used as delivery agents for increasing concentrations of 9.2s-RNA. Results of eight donors are shown as mean +/− SD. f – Done as described for (d) but polyethylenimine (PEI) was used to deliver increasing concentrations of 3p-dsRNA. Results of six donors are shown as mean +/− SD. g – Done as described for (d) but 0.8 µg/ml of dAdT was used to stimulate the cells. Results show mean of IFN-α and IP-10 induction of six donors +/− SD.</p

Amplification by transfection tolerates anticoagulation with Hirudin but not Heparin or EDTA in the WBA.

<p>a – Whole blood was anticoagulated with Heparin, EDTA or Hirudin and stimulated with 0.8 µg/ml B-type (2006) or C-type (M362) CpG-oligonucleotide or C20-DNA (negative control) in complex with DOTAP. After 48 h supernatant was analyzed for IFN-α induction by ELISA. Results of at least six donors are shown as mean +/− SD. b – Done as described for (a) but additional polyethylenimine (PEI) was used as delivery agent. Results of at least four donors are shown as mean +/− SD. c – Done as described for (a) but Protamine (Prot) and poly-L-Arginine (pArg) were used as delivery agents in complex with increasing concentrations of DNA as indicated. Results of at least eight donors are shown as mean +/− SD. d – Whole blood was anticoagulated with Hirudin and stimulated with 0.8 µg/ml CpG 2006 or CpC 2006 (CpG replaced by CpC sequences), both delivered with DOTAP. C20-DNA served as negative control. After 48 h supernatant was analyzed for IFN-α by ELISA. Results of eight donors are shown as mean +/− SD. e – CpG 2006 was incubated with DOTAP or pArg with or without addition of Heparin or Hirudin and digested with DNase before gel electrophoresis.</p

Comparison of cytokines induced by LPS and R848 in PBMC and in a human WBA.

<p>a – PBMC were incubated in 96 well with increasing concentrations of LPS. After 48 h supernatant was analyzed for TNF-α and IL-6 using ELISA. Results of eight donors are shown as mean +/− SD. b–c – Whole blood anticoagulated either with Heparin, EDTA or Hirudin was incubated in 96 wells with increasing concentrations of LPS and after 48 h supernatant was checked for IL-6 (b), TNF-α (c). Mean of six donors is shown +/− SD. d – Done as described in (a) but R848 was used for stimulation and supernatant was analyzed for IFN-α, TNF-α and IL-6. Mean of eight donors (three for IFN-α) is shown +/− SD. e–g – Done as described for (b) but R848 was used for stimulation and IFN-α (e), TNF-α (f) and IL-6 (g) were measured in supernatant. Results of four (IFN-α) and six donors are shown as mean +/− SD.</p

The Hirudin anticoagulated WBA exhibits high reliability upon repeated testing of the same donor.

<p>Whole blood of three different donors (a–c) anticoagulated with Hirudin was stimulated twice with one week inbetween with 0.8 µg/ml CpG 2006 delivered with polyethylenimine (PEI) or DOTAP. C20-DNA served as negative control. After 48 h supernatant was analyzed for IFN-α by ELISA. Mean of duplicates +/− SD is shown for each timepoint.</p

The WBA simulates IFN-α induction by CpG-ODN in vivo more accurately than the PBMC assay.

<p>a – PBMC were stimulated with B-type (CpG 2006) and C-type (M362) CpG-oligonucleotides as indicated. C20-DNA served as a negative control (neg contr). After 48 h induction of IFN-α was analyzed using ELISA. b – Done as described for (a) but whole blood anticoagulated with Heparin, EDTA or Hirudin was used. c – Complete whole blood or whole blood in which plasma was replaced by medium or medium+albumin was anticoagulated with Hirudin and stimulated with B-type (CpG 2006) and C-type (CpG M362) CpG-oligonucleotides or C20-DNA (neg contr) as indicated. After 48 h supernatant was analyzed for IFN-α concentration. Graph shows percentage of induced IFN-α compared to CpG M362 (induced IFN-α concentrations: 1 µg/ml CpG M362: 1697 pg/ml; 5 µg/ml CpG M362: 3627 pg/ml). Results of at least six donors are shown as mean +/− SD in (a–c).</p

RIG-I Detects Triphosphorylated RNA of <i>Listeria monocytogenes</i> during Infection in Non-Immune Cells

<div><p>The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as <i>Listeria</i>, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to <i>Listeria</i> DNA or <i>Listeria</i>-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of <i>Listeria</i> RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with <i>Listeria</i> as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent <i>Listeria</i> recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic <i>Listeria-</i>mediated type-I-IFN induction. Our results show that detection of <i>Listeria</i> RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.</p></div

Knockdown of RIG-I abrogates <i>L. monocytogenes</i>-induced type I IFN induction in epithelial but not in monocytic cells.

<p>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 <i>L. monocytogenes</i> or transfected with <i>L. monocytogenes</i> RNA (L.m. RNA), <i>L. monocytogenes</i> 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 <i>L. monocytogenes</i> 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.</p

Recognition of bacterial RNA or DNA varies for different cell types.

<p>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 <i>L. monocytogenes</i> RNA, <i>L. monocytogenes</i> 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 <i>L. monocytogenes</i> (L.M.) RNA. C, D, E and F: THP-1, A549, HepG2 and Colo205 cells were infected with wt and hly- <i>L. monocytogenes</i> 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.</p