title: Two phases of inflammatory mediator production defined by the study of IRAK2 and IRAK1 knock-in mice.
abstract: The roles of IL-1R-associated kinase (IRAK)2 and IRAK1 in cytokine production were investigated using immune cells from knock-in mice expressing the TNFR-associated factor 6 (TRAF6) binding-defective mutant IRAK2[E525A] or the catalytically inactive IRAK1[D359A] mutant. In bone marrow-derived macrophages (BMDMs), the IRAK2-TRAF6 interaction was required for the late (2-8 h) but not the early phase (0-2 h) of il6 and tnfa mRNA production, and hence for IL-6 and TNF-α secretion by TLR agonists that signal via MyD88. Loss of the IRAK2-TRAF6 interaction had little effect on the MyD88-dependent production of anti-inflammatory molecules produced during the early phase, such as Dual Specificity Phosphatase 1, and a modest effect on IL-10 secretion. The LPS/TLR4-stimulated production of il6 and tnfa mRNA and IL-6 and TNF-α secretion was hardly affected, because the Toll/IL-1R domain-containing adapter-inducing IFN-β (TRIF) signaling pathway was used instead of the IRAK2-TRAF6 interaction to sustain late-phase mRNA production. IRAK1 catalytic activity was not rate limiting for il6, tnfa, or il10 mRNA production or the secretion of these cytokines by BMDMs, but IFN-β mRNA induction by TLR7 and TLR9 agonists was greatly delayed in plasmacytoid dendritic cells (pDCs) from IRAK1[D359A] mice. In contrast, IFN-β mRNA production was little affected in pDCs from IRAK2[E525A] mice, but subsequent IFN-α mRNA production and IFN-α secretion were reduced. IFN-β and IFN-α production were abolished in pDCs from IRAK1[D359A] × IRAK2[E525A] double knock-in mice. Our results establish that the IRAK2-TRAF6 interaction is rate limiting for the late, but not the early phase of cytokine production in BMDM and pDCs, and that the IRAK2-TRAF6 interaction is needed to sustain IκB-inducing kinase β activity during prolonged activation of the MyD88 signaling network. [corrected]
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, United Kingdom.|IrsiCaixa, Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona 08916, Spain.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Pauls
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Sambit K
authors.initials: SK
authors.lastname: Nanda
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Hilary
authors.initials: H
authors.lastname: Smith
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Rachel
authors.initials: R
authors.lastname: Toth
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, United Kingdom.|Division of Cell Signaling and Immunology Unit, Sir James Black Centre, University of Dundee, DD1 5EH, United Kingdom.
authors.forename: J Simon C
authors.initials: JSC
authors.lastname: Arthur
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Philip
authors.initials: P
authors.lastname: Cohen
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis*; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D051550:I-kappa B Kinase / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D007249:Inflammation / Q000276:immunology / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases / Q000276:immunology / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D048029:TNF Receptor-Associated Factor 6 / Q000276:immunology / Q000378:metabolism; D014162:Transfection
chemical_list: D016207:Cytokines; D048029:TNF Receptor-Associated Factor 6; D053592:Interleukin-1 Receptor-Associated Kinases; C507769:Irak1 protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse
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