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Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1β production

Nature Immunology volume 15, pages 538–545 (2014)Cite this article

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Abstract

Double-stranded DNA (dsDNA) in the cytoplasm triggers the production of interleukin 1β (IL-1β) as an antiviral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune system adaptor CARD9. Transfection of dendritic cells with dsDNA or infection of dendritic cells with a DNA virus induced the formation of dsDNA-Rad50-CARD9 signaling complexes for activation of the transcription factor NF-κB and the generation of pro-IL-1β. Primary cells conditionally deficient in Rad50 or lacking CARD9 consequently exhibited defective DNA-induced production of IL-1β, and Card9−/− mice had impaired inflammatory responses after infection with a DNA virus in vivo. Our results define a cytosolic DNA-recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens.

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Figure 1: CARD9 interacts with Rad50.
Figure 2: CARD9 is recruited to cytoplasmic dsDNA-sensing Rad50 complexes.
Figure 3: CARD9 and Rad50 are essential for DNA-induced IL-1β production.
Figure 4: CARD9 controls dsDNA-mediated NF-κB activity.
Figure 5: Rad50-CARD9 interactions recruit Bcl-10 for IL-1β responses.
Figure 6: Recognition of infection with a DNA virus by Rad50-CARD9 complexes.
Figure 7: CARD9 controls DNA virus–induced immune responses in vivo.

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Acknowledgements

We thank M. Thome (University of Lausanne) for the antibody to CARD9; S. Essbauer (Bundeswehr Institute of Microbiology) for cowpox virus; and R. Ljapoci for technical assistance. Supported by the Bavarian Genome Research Network (A.C.M.), the Ludwig-Maximilians-Universität Excellence Initiative (42595-6 to A.C.M.), Deutsche Forschungsgemeinschaft (SFB684 to H.L. and J.R. and SFB1054 to J.R.), the US National Institutes of Health (U19AI083025 to K.-P.H. and GM56888 to J.H.J.P.) and the European Research Council (J.R.).

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Authors and Affiliations

  1. Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany

    Susanne Roth, Verena Laux & Jürgen Ruland

  2. Department Biology II and Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Martinsried, Germany

    Andrea Rottach & Heinrich Leonhardt

  3. Kinderklinik und Kinderpoliklinik im Dr. von Haunersches Kinderspital, Ludwig-Maximilians-Universität, Munich, Germany

    Amelie S Lotz-Havla, Søren W Gersting & Ania C Muntau

  4. Institut für Virologie, Klinikum rechts der Isar, Technische Universität München, and Helmholtz Zentrum München, German Research Centre for Environmental Health, Munich, Germany

    Andreas Muschaweckh & Ingo Drexler

  5. Department of Biochemistry, Gene Center and Center for integrated Protein Science Munich, Ludwig-Maximilians-Universität, Munich, Germany

    Karl-Peter Hopfner

  6. Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, USA

    Lei Jin

  7. Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Katelynd Vanness & John H J Petrini

  8. Institut für Virologie, Universitätsklinikum Düsseldorf, Heinrich Heine Universität, Düsseldorf, Germany

    Ingo Drexler

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  1. Susanne Roth
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Contributions

S.R., A.R., H.L. and J.R. designed the study; S.R., A.R., A.S.L.-H., V.L., A.M. and K.V. did the experiments; S.R., A.R., A.S.L.-H., S.W.G., A.C.M., K.-P.H., I.D., H.L. and J.R. analyzed the results; S.R., A.R. and A.S.L.-H. generated the figures; L.J. and J.H.J.P. provided reagents; and S.R. and J.R. wrote the paper.

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Correspondence to Jürgen Ruland.

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Integrated supplementary information

Supplementary Figure 1 MRN and Rad50-CARD9 complex formation at cytosolic dsDNA.

(a, b) BMDCs were transfected with dsDNA for 2 hours, stained with DAPI and anti-Rad50 and anti-Mre11 or anti-Nbs1 antibodies, and analyzed using confocal microscopy. The dsDNA/Rad50/Mre11 or dsDNA/Rad50/Nbs1 complexes (boxes) were also visualized at higher magnifications (Zoom). Scale bars represent 5 μm. The data are representative of at least three independent experiments analyzing at least 50 individual cells per experiment and assay point. (c-d) BMDCs were left untreated or transfected with poly(dG:dC) (2.5 μg/ml) for 2 hours and analyzed by confocal microscopy following immunofluorescence staining with DAPI and antibodies against Rad50 and CARD9. Localization of dsDNA, Rad50, and CARD9 in 100 cells per assay point was determined. (c) Percentages of cells containing Rad50-CARD9 aggregates compared to a diffuse localization of Rad50 and CARD9 are shown. (d) Percentages of WT and Card9-/- BMDCs containing cytosolic dsDNA at 2 hours after dsDNA transfection are demonstrated. (e) BMDCs from WT and Card9-/- mice were transfected with dsDNA, the relative number of cells containing Rad50 aggregates and homogenous Rad50 distribution, respectively, is shown. The data are representative of two independent experiments (c-e).

Supplementary Figure 2 ATM is not involved in dsDNA-induced IL-1b production.

BMDCs from WT and Atm-/- mice were transfected with dsDNA (1 - 4 μg/ml) of different origins for 16 hours or stimulated with LPS + ATP. The IL-1β concentrations in the supernatants were determined. The data are represented as the mean + SEM of triplicates of three independent experiments.

Supplementary Figure 3 CARD9 and Bcl-10 control pro-IL-1β synthesis.

BMDCs from mice of the indicated genotype were transfected with dsDNA (1 - 4 μg/ml) of different origins or stimulated with LPS, CpG, and curdlan plus ATP. The pro-IL-1β concentrations were determined in the cell lysates. The data are represented as the mean + SEM of triplicates. One representative of two independent experiments is shown. ND, not detectable.

Supplementary Figure 4 CARD9 regulates DNA-induced transcription of the genes encoding TNF and IL-6.

WT and Card9-/- BMDCs were transfected with poly(dG:dC) (2.5 μg/ml) for the indicated time and TNF (a) and IL-6 (b) transcript levels were measured by quantitative real-time PCR and normalized to β-actin mRNA levels. The data are shown as the mean + SEM of triplicates. One representative of two independent experiments is shown. **p < 0.01, ***p < 0.001, Student's t-test.

Supplementary Figure 5 cGAMP-STING signaling is independent of CARD9.

BMDCs from WT and Card9-/- (a), or WT and Tmem173-/- (b) mice were transfected with the STING activator cGAMP (4 μg/ml) for 6 hours. As control IFN-β production was induced via TLR9 ligation with CpG. IFN-β levels were measured in the supernatant. The data are represented as the mean + SEM of three (a), or two (b) independent experiments.

Supplementary Figure 6 CARD9 is crucial for RNA virus–induced IL-1β generation.

WT and Card9-/-BMDCs were infected with VSV at MOI 1. LPS, CpG, and curdlan plus ATP were used to investigate CARD9-independent and CARD9-dependent IL-1β production, respectively. The IL-1β concentrations in the supernatants were measured. The data are presented as the mean + SEM. One representative of three independent experiments is shown.

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Roth, S., Rottach, A., Lotz-Havla, A. et al. Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1β production. Nat Immunol 15, 538–545 (2014). https://doi.org/10.1038/ni.2888

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