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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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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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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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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DOI: https://doi.org/10.1038/ni.2378
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