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The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB

Nature Immunology volume 13, pages 823–831 (2012)Cite this article

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Abstract

Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3−/− mice. LPS-treated Nlrc3−/− macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3−/− mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

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Figure 1: NLRC3 inhibits MyD88- and TRAF6-dependent activation of NF-κB.
Figure 2: NLRC3 inhibits NF-κB by association with TRAF6.
Figure 3: NLRC3 promotes degradation of TRAF6 and prevents autoubiquitination of TRAF6.
Figure 4: Nlrc3−/− macrophages produce more proinflammatory cytokines in response to TLR agonists.
Figure 5: Nlrc3−/− macrophages produce more proinflammatory cytokines and TRAF6 in response to LPS.
Figure 6: Nlrc3−/− macrophages have more K63-linked ubiquitination of TRAF6 and activation of NF-κB.
Figure 7: Nlrc3−/− mice have a more severe reaction to sublethal challenge with LPS than do wild-type mice.

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Acknowledgements

We thank S. Ghosh (Columbia University, New York) for hemagglutinin-tagged MyD88 and Flag-tagged IRAK; Y. Xiong (University of North Carolina) for plasmids encoding hemagglutinin-tagged K48-linked ubiquitin and K63-linked ubiquitin; A. Baldwin (University of North Carolina) for Flag-tagged IKKα; and R. Thresher (University of North Carolina) for the targeting vector pOS. Supported by the US National Institutes of Health (R37-AI029564, U54-AI057157, U19-AI1077437 and T32-AR007416).

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Author notes

  1. Beckley K Davis, Eda K Holl, Zhengmao Ye, Adeeb H Rahman & Brian J Conti

    Present address: Present addresses: Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania, USA (B.K.D.); Duke Translational Research Institute, Duke University, Durham, North Carolina, USA (E.K.H.); HIV Drug Discovery Unit, Infectious Diseases Medicines Discovery & Development, GlaxoSmithKline, Research Triangle Park, North Carolina, USA (Z.Y.); Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York, USA (A.H.R.); and Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon, USA (B.J.C.).,

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Monika Schneider, Reid A Roberts, Eda K Holl, Zhengmao Ye, Brian J Conti & Jenny P-Y Ting

  2. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Albert G Zimmermann, Karen V Swanson, Haitao Wen, Beckley K Davis, Irving C Allen, Adeeb H Rahman, Timothy K Eitas & Jenny P-Y Ting

  3. School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Lu Zhang & Beverly H Koller

  4. Center for Translational Immunology and Institute for Inflammatory Diseases, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Jenny P-Y Ting

  5. Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Jenny P-Y Ting

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Contributions

M.S. did most of the experiments; J.P.-Y.T. supervised the project; M.S. and J.P.-Y.T. prepared the manuscript; A.G.Z. and B.H.K. generated the Nlrc3−/− mouse; L.Z., H.W. and T.K.E. contributed to biochemical analysis; K.V.S. did confocal assays; B.K.D. helped in design and creation of NLRC3 site mutants; I.C.A. contributed to experimental design and execution of mouse studies; B.J.C. designed NLRC3 retroviral vectors; R.A.R. did bioinformatics analysis and contributed to biochemical analysis; and A.H.R., E.K.H. and Z.Y. contributed to characterization of the Nlrc3−/− mouse.

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Correspondence to Jenny P-Y Ting.

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Schneider, M., Zimmermann, A., Roberts, R. et al. The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB. Nat Immunol 13, 823–831 (2012). https://doi.org/10.1038/ni.2378

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