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Author Notes:

Correspondence and requests for materials should be addressed to T.F.-A. (email: teresa.alnemri@jefferson.edu) or to E.S.A. (email: emad.alnemri@jefferson.edu).

S.K. and T.F.-A. contributed equally to this work.

T.F.-A. and E.S.A. conceived and designed the research and interpreted data.

T.F.-A., C.R., L.M. and E.S.A. performed all experiments.

S.K. performed all immunoblot assays for all experiments.

Y.W. provided Drp1fl/fl LysmCre+/− mice, C.D. and D.R.G. provided Mlkl−/− and Fadd/Mlkl−/− mice, L.R., W.K. and E.S.M. provided casp-8−/−/Ripk3−/−, Casp8−/−/Ripk1−/−/Ripk3−/−, Ripk1K45A/K45A and Ripk3K51A/K51A mice and commented on the manuscript.

A.O. and J.S. provided important reagents.

K.A.F. provided Nlrp3−/− mice and immortalized Ripk1−/− and Ripk+/+ fetal liver-derived macrophages.

J.Z. provided Ripk3−/− and Fadd−/−/Ripk3−/− mice.

E.S.A. supervised the research and wrote the manuscript.

We want to thank Vishva M. Dixit (Genentech) for the RIPK3-KO mice, James Murphy (The Walter and Eliza Hall Institute of Medical Research, Australia) for the MLKL-KO mice and Masatoshi Nomura (Kyushu University, Japan) and Ira Tabas (Columbia University) for the Drp1fl/fl mice.

We also thank Matthias Schnell (Thomas Jefferson University) for the VSV-GFP virus.

We also thank Maria Yolanda Covarrubias (Thomas Jefferson University) for assistance with the confocal microscopy, and Zhaozhao Jiang (University of Massachusetts Medical School) and Mao Yang (St Jude Children's Research Hospital) for assistance with preparation of mouse bones.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health (NIH).

The authors declare no competing financial interests.

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Research Funding:

Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number AR055398 to ESA.

C.R. was supported by NIH training grant T32 GM100836.

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • TOLL-LIKE RECEPTORS
  • CELL-DEATH
  • LISTERIA-MONOCYTOGENES
  • PROGRAMMED NECROSIS
  • PATTERN-RECOGNITION
  • SIGNALING PATHWAY
  • ASC PYROPTOSOME
  • RIP3 KINASE
  • K+ EFFLUX
  • APOPTOSIS
  • Inflammasome
  • Pattern recognition receptors
  • Post-translational modifications
  • Signal transduction

Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3

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Journal Title:

Nature Communications

Volume:

Volume 6

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Type of Work:

Article | Final Publisher PDF

Abstract:

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.

Copyright information:

© 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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