Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Apr 30;8(4):e62872.
doi: 10.1371/journal.pone.0062872. Print 2013.

RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells

Cristina Amparo Hagmann  1 , Anna Maria Herzner, Zeinab Abdullah, Thomas Zillinger, Christopher Jakobs, Christine Schuberth, Christoph Coch, Paul G Higgins, Hilmar Wisplinghoff, Winfried Barchet, Veit Hornung, Gunther Hartmann, Martin Schlee
Affiliations

RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells

Cristina Amparo Hagmann et al. PLoS One. .

Abstract

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 Listeria, 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 Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria 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 Listeria 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 Listeria 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 Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Bacterial RNA is recognized by human monocytes in a TLR-independent and 5′phosphorylation-dependent pathway.
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.
Figure 2
Figure 2. RNA of L. monocytogenes has access to the cytosol of the host cell during infection.
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).
Figure 3
Figure 3. Recognition of bacterial RNA or DNA varies for different cell types.
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.
Figure 4
Figure 4. Knockdown of RIG-I abrogates L. monocytogenes-induced type I IFN induction in epithelial but not in monocytic cells.
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chen G, Shaw MH, Kim YG, Nunez G (2009) NOD-like receptors: role in innate immunity and inflammatory disease. Annu Rev Pathol 4: 365–398. - PubMed
    1. Schlee M, Hornung V, Hartmann G (2006) siRNA and isRNA: two edges of one sword. Mol Ther 14: 463–470. - PubMed
    1. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, et al. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374: 546–549. - PubMed
    1. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408: 740–745. - PubMed
    1. Coch C, Busch N, Wimmenauer V, Hartmann E, Janke M, et al. (2009) Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA. J Leukoc Biol 86: 663–670. - PubMed

Publication types

MeSH terms

Grants and funding

This study was supported by Grants BMBF Biofuture 0311896, BMBF-grant 01KI0771 (www.bmbf.de/) to GH and from the Deutsche Forschungsgemeinschaft (www.dfg.de; SFB670 to MS, GH and VH, SFB704 to GH, SFB832 and KFO177 to GH). GH, WB and VH are members of the DFG Excellence Cluster ImmunoSensation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.