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

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.

References

  1. Paludan, S.R. & Bowie, A.G. Immune sensing of DNA. Immunity 38, 870–880 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

    CAS  PubMed  Google Scholar 

  3. Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).

    CAS  PubMed  Google Scholar 

  4. Goubau, D., Deddouche, S. & Reis, E.S.C. Cytosolic sensing of viruses. Immunity 38, 855–869 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Ishikawa, H., Ma, Z. & Barber, G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhang, Z. et al. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat. Immunol. 12, 959–965 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Epperson, M.L., Lee, C.A. & Fremont, D.H. Subversion of cytokine networks by virally encoded decoy receptors. Immunol. Rev. 250, 199–215 (2012).

    PubMed  PubMed Central  Google Scholar 

  8. Smith, G.L. et al. Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity. J. Gen. Virol. 94, 2367–2392 (2013).

    CAS  PubMed  Google Scholar 

  9. Staib, C., Kisling, S., Erfle, V. & Sutter, G. Inactivation of the viral interleukin 1beta receptor improves CD8+ T-cell memory responses elicited upon immunization with modified vaccinia virus Ankara. J. Gen. Virol. 86, 1997–2006 (2005).

    CAS  PubMed  Google Scholar 

  10. Zimmerling, S., Waibler, Z., Resch, T., Sutter, G. & Schwantes, A. Interleukin-1β receptor expressed by modified vaccinia virus Ankara interferes with interleukin-1β activity produced in various virus-infected antigen-presenting cells. Virol. J. 10, 34 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Dinarello, C.A., Simon, A. & van der Meer, J.W. Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases. Nat. Rev. Drug Discov. 11, 633–652 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Vallabhapurapu, S. & Karin, M. Regulation and function of NF-κB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733 (2009).

    CAS  PubMed  Google Scholar 

  13. Roth, S. & Ruland, J. Caspase recruitment domain-containing protein 9 signaling in innate immunity and inflammation. Trends Immunol. 34, 243–250 (2013).

    CAS  PubMed  Google Scholar 

  14. Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805–820 (2010).

    CAS  PubMed  Google Scholar 

  15. Gross, O. et al. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature 442, 651–656 (2006).

    CAS  PubMed  Google Scholar 

  16. Hara, H. et al. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat. Immunol. 8, 619–629 (2007).

    CAS  PubMed  Google Scholar 

  17. Hsu, Y.M. et al. The adaptor protein CARD9 is required for innate immune responses to intracellular pathogens. Nat. Immunol. 8, 198–205 (2007).

    CAS  PubMed  Google Scholar 

  18. Poeck, H. et al. Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1β production. Nat. Immunol. 11, 63–69 (2010).

    CAS  PubMed  Google Scholar 

  19. Strasser, D. et al. Syk kinase-coupled C-type lectin receptors engage protein kinase C-δ to elicit Card9 adaptor-mediated innate immunity. Immunity 36, 32–42 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Stracker, T.H. & Petrini, J.H. The MRE11 complex: starting from the ends. Nat. Rev. Mol. Cell Biol. 12, 90–103 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Gersting, S.W., Lotz-Havla, A.S. & Muntau, A.C. Bioluminescence resonance energy transfer: an emerging tool for the detection of protein-protein interaction in living cells. Methods Mol. Biol. 815, 253–263 (2012).

    CAS  PubMed  Google Scholar 

  22. Maser, R.S., Monsen, K.J., Nelms, B.E. & Petrini, J.H. hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol. Cell. Biol. 17, 6087–6096 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Goodridge, H.S. et al. Differential use of CARD9 by dectin-1 in macrophages and dendritic cells. J. Immunol. 182, 1146–1154 (2009).

    CAS  PubMed  Google Scholar 

  24. Adelman, C.A., De, S. & Petrini, J.H. Rad50 is dispensable for the maintenance and viability of postmitotic tissues. Mol. Cell. Biol. 29, 483–492 (2009).

    CAS  PubMed  Google Scholar 

  25. Luo, G. et al. Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation. Proc. Natl. Acad. Sci. USA 96, 7376–7381 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Barlow, C. et al. Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 86, 159–171 (1996).

    CAS  PubMed  Google Scholar 

  27. Roberts, K.L. & Smith, G.L. Vaccinia virus morphogenesis and dissemination. Trends Microbiol. 16, 472–479 (2008).

    CAS  PubMed  Google Scholar 

  28. Abdullah, Z. et al. RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids. EMBO J. 31, 4153–4164 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Kok, K.H. et al. The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response. Cell Host Microbe 9, 299–309 (2011).

    CAS  PubMed  Google Scholar 

  30. Li, X.D. et al. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394 (2013).

    CAS  PubMed  Google Scholar 

  31. Fernandes-Alnemri, T. et al. The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat. Immunol. 11, 385–393 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Rathinam, V.A. et al. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat. Immunol. 11, 395–402 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Carney, J.P. et al. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93, 477–486 (1998).

    CAS  PubMed  Google Scholar 

  34. Lilley, C.E., Schwartz, R.A. & Weitzman, M.D. Using or abusing: viruses and the cellular DNA damage response. Trends Microbiol. 15, 119–126 (2007).

    CAS  PubMed  Google Scholar 

  35. Weitzman, M.D., Carson, C.T., Schwartz, R.A. & Lilley, C.E. Interactions of viruses with the cellular DNA repair machinery. DNA Repair (Amst.) 3, 1165–1173 (2004).

    CAS  Google Scholar 

  36. Kondo, T. et al. DNA damage sensor MRE11 recognizes cytosolic double-stranded DNA and induces type I interferon by regulating STING trafficking. Proc. Natl. Acad. Sci. USA 110, 2969–2974 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Ferguson, B.J., Mansur, D.S., Peters, N.E., Ren, H. & Smith, G.L. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. Elife 1, e00047 (2012).

    PubMed  PubMed Central  Google Scholar 

  38. Zhang, X. et al. Cutting edge: Ku70 is a novel cytosolic DNA sensor that induces type III rather than type I IFN. J. Immunol. 186, 4541–4545 (2011).

    CAS  PubMed  Google Scholar 

  39. Saiga, H. et al. Critical role of AIM2 in Mycobacterium tuberculosis infection. Int. Immunol. 24, 637–644 (2012).

    CAS  PubMed  Google Scholar 

  40. Alcamí, A. & Smith, G.L. A mechanism for the inhibition of fever by a virus. Proc. Natl. Acad. Sci. USA 93, 11029–11034 (1996).

    PubMed  PubMed Central  Google Scholar 

  41. Stracker, T.H., Carson, C.T. & Weitzman, M.D. Adenovirus oncoproteins inactivate the Mre11-Rad50–NBS1 DNA repair complex. Nature 418, 348–352 (2002).

    CAS  PubMed  Google Scholar 

  42. Boswell, J.M., Yui, M.A., Burt, D.W. & Kelley, V.E. Increased tumor necrosis factor and IL-1β gene expression in the kidneys of mice with lupus nephritis. J. Immunol. 141, 3050–3054 (1988).

    CAS  PubMed  Google Scholar 

  43. Dombrowski, Y. et al. Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions. Sci. Transl. Med. 3, 82ra38 (2011).

    PubMed  PubMed Central  Google Scholar 

  44. Popovic, K. et al. Increased expression of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 in skin lesions of patients with lupus erythematosus. Arthritis Rheum. 52, 3639–3645 (2005).

    CAS  PubMed  Google Scholar 

  45. Ruland, J. et al. Bcl10 is a positive regulator of antigen receptor-induced activation of NF-κB and neural tube closure. Cell 104, 33–42 (2001).

    CAS  PubMed  Google Scholar 

  46. Jin, L. et al. MPYS is required for IFN response factor 3 activation and type I IFN production in the response of cultured phagocytes to bacterial second messengers cyclic-di-AMP and cyclic-di-GMP. J. Immunol. 187, 2595–2601 (2011).

    CAS  PubMed  Google Scholar 

  47. Mariathasan, S. et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213–218 (2004).

    CAS  PubMed  Google Scholar 

  48. Gasteiger, G., Kastenmuller, W., Ljapoci, R., Sutter, G. & Drexler, I. Cross-priming of cytotoxic T cells dictates antigen requisites for modified vaccinia virus Ankara vector vaccines. J. Virol. 81, 11925–11936 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Bürckstümmer, T. et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat. Immunol. 10, 266–272 (2009).

    PubMed  Google Scholar 

  50. Cremer, M. et al. Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes. Methods Mol. Biol. 463, 205–239 (2008).

    CAS  PubMed  Google Scholar 

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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

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.

Corresponding author

Correspondence to Jürgen Ruland.

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The authors declare no competing financial interests.

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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